Development of New Tools for Study of Tumor Microenvironment

Solid tumors have a microenvironment that is inherently acidic and hypoxic. Hypoxia is caused by leaky blood vessels and large diffusion distances from cells to them. It is heterogeneous throughout the tumor and while all solid tumors are hypoxic to a degree, it is difficult to predict invasiveness based on it. However, acidity is a near ubiquitous characteristic of tumors with more aggressive tumors producing greater acidity. It is important to measure pH in diseased tissue with accuracy and precision, since acidity is associated with the development of various pathological states including tumors. In this work we focus on the acidosis aspect of the tumor microenvironment by describing the development of pHLIP  (pH (Low) Insertion Peptides) targeting based tools that are capable of imaging the pH of a tumor microenvironment. pHLIP was chosen as a targeting vehicle because of its pH dependent insertion mechanism that allows it to effectively target acidic tissues,

since acidity is associated with the development of various pathological states including tumors. In this work we focus on the acidosis aspect of the tumor microenvironment by describing the development of pHLIP  (pH (Low) Insertion Peptides) targeting based tools that are capable of imaging the pH of a tumor microenvironment. pHLIP was chosen as a targeting vehicle because of its pH dependent insertion mechanism that allows it to effectively target acidic tissues, including tumors.
We used pHLIP® to study the roles of carboxyl groups in transmembrane (TM) peptide insertion. pHLIP binds to the surface of a lipid bilayer as a disordered peptide at neutral pH; when the pH is lowered, it inserts across the membrane to form a TM helix. Peptide insertion is reversed when the pH is raised above the characteristic pK a (6.0). A key event that facilitates membrane insertion is the protonation of aspartic acid (Asp) and/or glutamic acid (Glu) residues, since their negatively charged side chains hinder membrane insertion at neutral pH. In order to gain mechanistic understanding, we studied the membrane insertion and exit of a series of pHLIP variants where the four Asp residues were sequentially mutated to nonacidic residues, including histidine (His). Our results show that the presence of His residues does not prevent the pH-dependent peptide membrane insertion at ∼ pH 4 driven by the protonation of carboxyl groups at the inserting end of the peptide. We expect that our understanding will be used to improve the targeting of acidic diseased tissue by pHLIP.
Looking from the lipid bilayer's perspective, small angle x-ray scattering studies showed membrane thinning by 18% induced by insertion of short-pHLIP (truncated version of pH Low Insertion Peptide) into bilayer. Thinning allows to reduce stress on membrane associated with negative hydrophobic mismatch. Also we observed 12% of membrane thinning when long-pHLIP partitions into outer leaflet of bilayer at high pH adopting coil conformations. The long-pHLIP at high pH creates an asymmetric inclusion in the bilayer, which results in increase of tension leading to the bilayer thinning. The tension and thinning is released when long-pHLIP inserts into bilayer as a transmembrane helix at low pH.
The first tool developed is a new 64 Cu-pHLIP peptide for targeting, imaging and quantifying acidic tumors by positron emission tomography, and our findings reveal utility in assessing prostate tumors. The new pHLIP version limits indiscriminate healthy tissue binding, and we demonstrate its targeting of extracellular acidification in three different prostate cancer models, each with different vascularization and acidextruding protein carbonic anhydrase IX (CAIX) expression. We then describe the tumor distribution of this radiotracer ex vivo, in association with blood perfusion and known biomarkers of acidity such as hypoxia, lactate dehydrogenase A and CAIX. We find that the new probe reveals metabolic variations between and within tumors, and discriminates between necrotic and living tumor areas.
The second tool introduced is a novel approach of extracellular pH measurements at the surface of cells, which is based on the use of a pH-sensitive fluorescent dye SNARF conjugated to a pH Low Insertion Peptide (WT-pHLIP), which targets plasma v ACKNOWLEDGMENTS First I would like to thank my advisor Dr Oleg Andreev and Yana Reshetnyak for their support throughout this process. Honestly I do not know where I would be without them. They had faith in me even before they knew me. They gave me oppurtunities that I never deserved. They were patient with me throughout all my mistakes (and there was a lot of them) and made sure that I would never fail. Ultimately I feel privledged to have had researchers of their caliber mentor me.
I thank my parents for their relentless support of me. They always made sure that I had the best medical care and that I was taken care of. It didn't matter if I called them up falling apart, they would listen to me and help me get through it. I will never forget the constant encouragement that they give me.
I am forever grateful to my wife Leann Anderson for always supporting me and moving far from home with me just so that I could go to graduate school. She always made coming home pleasurable and is truly the only person who understands me.
Also, for not only condoning, but contributing to my cat loving ways.
To my fur baby, Bella, for making bad days better with your cuddles.
A big thank you to my Uncle Dick for always challenging me to think, supporting me and exposing me to different experiences.
Thank you Grandma Reedy, while she has passed on, my grandma always encouraged me in my studies and never doubted me.
I would like to thank Mr. Jonathon Zinnel. While I am sure that he will never know how much he helped me, it was his faith in me at the start of 8 th grade that completely changed the course of my academic career.
vi Also thank you Mr. Maurice Green for giving me so much of your time, energy and defending me at the end of high school. He was the reason that I learned to love and appreciate physics.
I am grateful to Dr. Bartley Cardon for his support throughout undergraduate school.

Abstract
We have used pHLIP® [pH (low) insertion peptide] to study the roles of carboxyl groups in transmembrane (TM) peptide insertion. pHLIP binds to the surface of a lipid bilayer as a disordered peptide at neutral pH; when the pH is lowered, it inserts across the membrane to form a TM helix. Peptide insertion is reversed when the pH is raised above the characteristic pK a (6.0). A key event that facilitates membrane insertion is the protonation of aspartic acid (Asp) and/or glutamic acid (Glu) residues, since their negatively charged side chains hinder membrane insertion at neutral pH. In order to gain mechanistic understanding, we studied the membrane insertion and exit of a series of pHLIP variants where the four Asp residues were sequentially mutated to nonacidic residues, including histidine (His). Our results show that the presence of His residues does not prevent the pH-dependent peptide membrane insertion at ∼ pH 4 driven by the protonation of carboxyl groups at the inserting end of the peptide. A further pH drop leads to the protonation of His residues in the TM part of the peptide, which induces peptide exit from the bilayer. We also find that the number of ionizable residues that undergo a change in protonation during membrane insertion correlates with the pH-dependent insertion into the lipid bilayer and exit from the lipid bilayer, and that cooperativity increases with their number. We expect that our understanding will be used to improve the targeting of acidic diseased tissue by pHLIP.

Introduction
Extracellular acidification is a hallmark of different pathologies, including cancer, inflammation, ischemic stroke, and atherosclerotic plaques. Acidosis might be a useful biomarker for diagnosis or treatment if means can be found to target tissue acidity. We have found that a peptide derived from helix C of bacteriorhodopsin, 1 named pHLIP® [pH (low) insertion peptide], is capable of targeting acidic tissues and inserting into the cell plasma membrane. 2 pHLIP is able to target mouse tumors in vivo with high specificity, 2 opening the possibility of its use for cancer imaging. Additionally, pHLIP has a promising therapeutic potential, as it is able to translocate cell-impermeable cargo molecules, such as organic dyes, peptides, peptide nucleic acids, and toxins, across the plasma membrane into the cytoplasm of tumor cells. 2  if the solution pH is lowered, pHLIP inserts to form a transmembrane (TM) α-helix (state III). The insertion is fully reversible and unidirectional, with the C-terminus being translocated across the membrane. 3 The pK a of peptide insertion into lipid bilayers is 6.0, and the energy difference between the attached state and the inserted state is 1.8 kcal/mol at 37 °C. 4 The pHLIP sequence is relatively rich in acidic residues (Table 1). At neutral pH, the combined negative charges of these residues, together with the carboxy terminus, constitute a large energetic barrier to pHLIP insertion across the membrane. The estimated energetic cost of the transfer of a single aspartic acid residue from water to the hydrophobic core of the membrane is unfavorable by 3.6 kcal/mol for the unprotonated (negatively charged) state, but only by 0.4 kcal/mol for the protonated (noncharged) state. 5 Simultaneously moving four charged Asp residues, one Glu residue, and the carboxy terminus into the membrane would cost 21.6 kcal/mol, assuming 3.6 kcal/mol for each carboxyl group, and peptide partitioning into the membrane at equilibrium would be about 1:10 16 . Thus, for pHLIP to be able to insert into membranes, protonation of a large fraction of the acidic residues can be expected, and knowledge of the protonation pattern of the acidic residues of pHLIP is an essential part of understanding the molecular mechanism of the membrane insertion process for any peptide containing carboxyl groups. Two classes of carboxyl groups are of interest: those that remain buried in the membrane after pHLIP is inserted into the membrane and those that traverse the hydrophobic core of the membrane during insertion. 6 Accordingly, we have studied both the pH-driven membrane insertion and the exit process for a series of peptides where the key aspartic acid residues are sequentially mutated.

Results
Previous studies in our laboratories revealed that sequence variations in the TM region of pHLIP can disrupt the delicate balance that preserves its water solubility. For example, a simultaneous change in the two aspartic acid residues at positions 14 and 25 to the homologous glutamic acid (Asp14/25Glu) resulted in a loss of pH-dependent membrane insertion due to aggregation of the peptide in aqueous solution 7 (we have recently developed new pHLIP variants with several Glu residues, which preserve pHdependent properties; unpublished data). In order to reduce the likelihood that the introduced variations in the peptides used in this work could cause aggregation, we decided to follow a dual strategy to increase their water solubility: (i) we added an Asp tag to the N-terminus (noninserting end) to increase the number of charges in the molecule, which typically improves the solubility of hydrophobic peptides 8 and 9 ; this resulted in the replacement of the N-terminal sequence AAEQ with DDDED (Table 1); and (ii) we used the TANGO algorithm 10 to define the region of the pHLIP sequence with the highest aggregation tendency and found this to be the stretch from residue 21 to residue 30 (coinciding with the most hydrophobic region of the peptide).
We then mutated Leu26 to Gly, which greatly reduced the predicted aggregation tendency.
We incorporated these modifications into a series of pHLIP variants, where four aspartic acid residues were sequentially mutated to nonacidic polar residues. The aspartic acid residues at the C-terminus of the peptide that transitorily traverse the core of the membrane upon insertion (Asp31 and Asp33) were replaced with polar but uncharged asparagine residues. On the other hand, for the Asp residues that are located in the core of the membrane after insertion (in positions 14 and 25), histidine was chosen as the replacement residue, as it is expected to be partially charged at neutral pH (thus improving water solubility) while being only slightly polar in its uncharged state (the transfer energies from water to the bilayer interior are 0.43 and 0.11 kcal/mol for the neutral forms of Asp and His, 5 respectively) so that the insertion properties of pHLIP may not be altered. The peptides were named D0-D3 according to the number of aspartic acid residues present in the regions of interest (TM and Cterminus; the positively charged N-terminus is not expected to interact with the membrane). For the variants with three aspartic acids, two alternatives were studied: one that kept Asp14 (D3a peptide) and the other that kept Asp25 (D3b peptide).
We conducted experiments to test the state of the variants in solution, where pHLIP is largely found as an unstructured monomer. 11 Sedimentation velocity experiments were conducted to determine the oligomerization state of the different peptide variants in aqueous buffer. Previous analysis of wild-type (wt) pHLIP (at 7 μM in 10 mM phosphate buffer and 100 mM NaCl, pH 8) 11 showed that pHLIP is mostly monomeric, but a small oligomer population is observed (∼ 6%). We performed our sedimentation velocity experiments under the same conditions, but without NaCl in the solution. For each peptide, we observed a peak with a sedimentation coefficient of 0.72 ± 0.12 S (Table 2 and Fig. 1 (Fig. 3). The CD signature of the pHLIP membrane insertion process consists of the appearance of the characteristic signals associated with the formation of α-helix: minima at 208 and 222 nm and positive ellipticity at 190 nm. Both D3 variants showed spectral changes very similar to those observed for wt upon acidification. Thus, we concluded that replacement of one of the Asp residues in the TM region of the peptide does not lead to changes in the peptide's ability to interact with the membrane in a pH-dependent manner.
The D2 variant, where both Asp residues are replaced by His residues, also demonstrates a pH-dependent membrane interaction. However, the spectral pattern is slightly different from those for wt and D3 variants: the fluorescence intensity of D2 in the presence of POPC decreases in the pH range 8-6, with no significant changes in the spectral maximum at pH 8-7 and with a small shift to lower wavelengths at pH 6 ( Fig. S1). The amount of the helical structure of D2 at neutral pH is slightly higher than those of wt and D3 (Fig. 2 and Table 2), while no change is seen in the pH range 8-6. As an explanation, we suggest that D2 partitions somewhat more deeply into the membrane lipid bilayer than wt and D3 at neutral pH values, since His residues are expected to be only partially charged at neutral pH values, enhancing the hydrophobicity of the peptide TM and its affinity for the lipid bilayer. The decrease in fluorescence signal in the pH range 8-6 might be attributed to the partial quenching of emission of at least one of the Trp residues by one of the partially protonated His residues. At the same time, at neutral pH values, the peptide C-terminus containing four negative charges (two Asp, one Glu, and the C-terminus) does not partition into the membrane, keeping the peptide at the membrane surface. A further drop of the pH to pH 3-4 is associated with a fluorescence spectral maximum blueshift, an increase in fluorescence intensity (Fig. 2), and the appearance of a more pronounced negative band at 222 nm on CD spectra (Fig. 3), which is usually an indication of peptide insertion into the bilayer. 1 Reduction of pH leads to the protonation of negatively charged groups at the C-terminus and peptide insertion into the membrane. At the same time, we expect that protonation of His residues at low pH should occur; this might lead to the peptide's exit from the lipid bilayer or, alternatively, the formation of a pore channel in the lipid bilayer, where positively charged His residues would be pointed toward the channel. Calcein encapsulation control experiments that rule out the formation of pores in the membrane by the D2 and D3 peptides were performed ( Fig. S2). Thus, most probably, the pK a for the protonation of His is shifted to very low pH values when it is embedded in a lipid bilayer. We carried out fluorescence pH titrations to compare the behaviors of D2 and wt peptides at pH values lower than 3.5 ( Fig. S3). While no fluorescence change was detected for wt at acidic pH values, we observed that an additional process was present for D2 (with an apparent pK a of 2.5), characterized by a fluorescence decrease and a redshift of the spectral maximum, which might be associated with peptide exit from the lipid bilayer.
To establish the orientation of each helix in the membrane, we performed oriented circular dichroism (OCD) measurements in which the light beam is oriented perpendicular to the planes of a stack of oriented lipid bilayers containing the peptides of interest. Theoretical calculations and experimental data indicate that helices oriented with axes parallel with the membrane surface (perpendicular to the incident light) give CD signals distinctly different from those of helices oriented across the bilayer (parallel with the incident light). 12, 13 and 14 In the range of 190-240 nm, the peptide CD spectrum is dominated by π-π* and n-π* transitions. 15 The π-π* transition in a helix splits into three components, one of which gives rise to a negative Gaussian band near 205 nm, with its electric transition dipole parallel with the helical axis.
When the incident light propagates parallel with the helical axis, the electric field vector is orthogonal to the 205-nm π-π* dipole transition, and there is no interaction between the electromagnetic wave and the dipole, leading to the disappearance of the negative band at 205 nm in a CD spectrum. Thus, when the supported bilayers are oriented perpendicular to the light propagation, a helix with a TM orientation will have a CD spectrum that contains a positive 190-nm band and a negative 225-nm band. If the helix adopts a membrane surface orientation on the supported bilayer, then all transitions are seen, and the OCD spectrum is the same as for a peptide CD spectrum in solution, with randomly oriented helices. Our data clearly indicate that D2 adopts a TM orientation at pH 3.5-4.5, while increasing the pH leads to peptide exit and the appearance of a membrane surface orientation of the helix (Fig. 4). The OCD spectrum at pH 1.9 does not correspond to a TM helix. Thus, we conclude that the pK a of both or at least one of the His residues is significantly shifted from 6.3-6.9 16 to a lower value (2.5) due to their location at the bilayer interface in state II, emphasizing the important influence of bilayer surface properties on the pK values of dissociating groups in interacting peptides. A similar trend was previously observed for peptides that insert into membranes via the deprotonation of His residues, 17 and 18 although the magnitude of the pK a shift was smaller. However, large changes in pK a are typically observed when the side chains are in different environments, as the protonation of titratable amino acids depends on the dielectric properties of their environment. 19 A fitting example of large pK a changes is found in the native environment of pHLIP, bacteriorhodopsin, where Asp14 and Asp25 have pK a values of 7.5 and > 9, respectively, 20 significantly higher than the pK a values of 3.7-4.0 found for fully solvated aspartic acid side chains. 16 D1 has one less Asp residue at the C-terminus than D2. The slightly larger blueshift of fluorescence emission ( To study the magnitude and directionality of the membrane insertion of the peptides, we used a biotin-avidin binding assay. A biotin moiety was attached to the C-terminus of each peptide. The level of binding to avidin was measured, and the protection of the biotin molecule from avidin interaction was used to assess the translocation of the peptide C-terminus into the liposome interior. The biotin moiety was linked to the Cterminal Cys of the peptides via a long polar polyethylene glycol (PEG) linker. The linker has a double purpose. It facilitates biotin access to the avidin binding site andmore critically for our experiments-helps to delineate between an intraliposomal location and an extraliposomal location of the biotin, since the polarity of the moiety makes a location inside the hydrophobic region of the bilayer unlikely. We quantified the amount of biotin that binds to avidin molecules present exclusively outside the liposomes (see Materials and Methods for details). We did not detect avidin binding to biotin for the D2 peptide at low pH ( Fig. 5a) due to the biotin translocation across the membrane, which complements our data (suggesting complete insertion of this peptide across the lipid bilayer) and confirms that the directionality of insertion is the same as for wt. Only partial translocation and no translocation of biotin across the membrane were seen for D1 and D0, respectively (Fig. 5a), in agreement with our results indicating partial (or tilted) insertion and no insertion into the lipid bilayer of D1 and D0, respectively. Additionally, the translocation of biotin (which can be considered as a cargo) across the membrane does not appear to significantly hinder the membrane insertion of the peptides. This might be explained by its small size (526 Da) and its moderate polarity (logP = − 1.4; see Materials and Methods for details), which are both well within the range of cargo properties that pHLIP has been reported to effectively translocate. 21 However, as the biotin assay used here is responsive to changes in the level of binding to avidin present outside of the liposomes, we cannot rule out the possible influences of different processes such as peptide aggregation, although we have no reason to suspect them.
How does the number of carboxyl groups affect the pK and cooperativity of insertion?
We monitored the pH-induced changes in the position of the fluorescence emission maximum of the peptides, which provide details about peptide insertion into the lipid bilayer, in the presence of POPC (Fig. 6). A plot of the positions of the spectral maxima follows a sigmoid behavior as a function of pH, corresponding to the transition between the interfacial state and the inserted state for all variants (except for D0). Fitting the experimental data provides the two main parameters that describe the insertion process: pK a and cooperativity (m). The pK a of membrane insertion obtained for wt pHLIP is 5.94 ± 0.09, which is in agreement with previous reports. 1 and 7 For the different variants, shifts of the pK a to lower values (∼ 5.2) were detected (Fig. 7a).
The reason for this decrease is unclear, but it might be related to the lower number of aspartic residues or to the presence of histidines in the TM region of the pHLIP variants. We do not think that the N-terminal DDDED sequence will influence the pK a values of the peptides in our study, since its polarity should preclude hydrophobic interaction with the lipid bilayer; thus, it is not expected to be involved in the insertion process. However, we cannot rule out that it might reduce the overall membrane affinity of the peptide. While the pK a values for the variants changed very little, we observed a gradual decrease in the cooperativity of the insertion process (m parameter) for peptides with fewer Asp residues, as the titration occurred progressively over a wider pH range (∼ 1 pH unit for wt and ∼ 2 pH units for D1) (Figs. 6 and 7b). Our data indicate that the cooperativity of insertion is linked to the number of protonatable residues. Cooperativity and pK a might also respond to the position of protonatable groups in the peptide sequences and their proximity to each other. When pHLIP is at the surface of the vesicle and the pH is lowered, the protonation of one Asp residue might facilitate the protonation of other protonatable residues, shifting their pK a values. The protonation of the first Asp residue might induce partial insertion of the peptide into the membrane. In this scenario, the protonation of the neighboring Asp residues would be energetically favored to shield the negative charge (i.e., the pK a value of the neighboring Asp is shifted to higher values in a more hydrophobic environment) and then a positive feedback would be established, triggering membrane insertion.
How do the number and the location of Asp residues affect peptide exit from the membrane? The CD and fluorescence changes associated with wt pHLIP lipid insertion at acidic pH are completely reversible. 11 Here we also followed changes in the CD and fluorescence signals and in the reversibility of biotin translocation across the membrane. The ellipticity increase associated with each peptide insertion into the membrane was found to be essentially reversible for wt and D3b (Fig. 3, broken blue lines overlap with continuous blue lines), while for D3a, D2, and D1, the reversibility was only partial. Since changes in the CD signal upon acidification for D2-D0 are less pronounced than those for wt and D3, the reversibility of the D2-D0 membrane insertion was also assessed by changes in the fluorescence signal (Fig. S4). It is interesting to note the different levels of reversibility of the two D3 peptides: the insertion process is significantly more reversible in D3b (90%) than in D3a (70%) ( Fig. 5b), suggesting nonequivalence of the two buried positions. We observed an overall linear relationship between the number of aspartic acid residues interacting with the membrane and the degree of α-helix formation reversibility (Fig. 5b). The results obtained for the reversibility of the biotin translocation (exit process) were also in agreement (Fig. 5b).
An important consideration in the interpretation of the exit data is the time course of equilibration of the pH inside the liposomes, so we encapsulated the membrane-

Discussion
We have previously observed that even conservative changes in the pHLIP sequence can lead to peptide aggregation in solution at neutral pH. 7 Our results show that all the peptides in this study are soluble in solution, being essentially monomeric (the addition of a D-tag at the N-terminus and the L26G mutation appear to favor peptide solubility). Spectral data obtained with D3-D0 peptides indicate that the lower is the number of negatively charged groups in the peptide sequence, the deeper are the peptide partitions into a lipid bilayer and the greater is the helicity. At the same time, TM orientation (at least for the D3-D2 peptides) requires protonation of the Asp/Glu residues and the terminal carboxyl group at the C-terminus, which can readily go across a membrane in its noncharged form. We confirmed our previous finding 2 suggesting that TM Asp residues are not essential for peptide insertion. Interestingly, we have observed here that membrane insertion upon acidification occurs in our peptides in the presence of two His residues in the predicted TM region. Histidines have been used in the past to drive the insertion of peptides into membranes at neutral pH values. 17  We conclude that protonation of negatively charged residues located in the TM or in the C-terminal inserting end must occur in order to preserve the pH-dependent ability of pHLIP to interact with the membrane. These residues act as switches for pHLIP membrane insertion, as the negative charges of their side chains block membrane insertion. Acidification causes the protonation of these side chains, resulting in an increase in the overall hydrophobicity of the peptide, which leads to TM helix formation, shielding the hydrophobic residues of pHLIP from water molecules. When the pH is raised to near neutrality, the negatively charged state of the carboxyl groups is again favored, decreasing the peptide hydrophobicity and resulting in exit from the TM position. Peptide exit from the lipid bilayer is completed when deprotonation of Asp/Glu residues located in the hydrophobic core of the membrane occurs and the TM helix is destabilized. Thus, pHLIP variants where Asp14/Asp25 were replaced by Glu, with a higher pK a (pK a = 6.5), 7 might be more effective for targeting tumors with higher pH e values. Our present results suggest that the number of Asp residues in the TM region can also modulate the pK a value. Thus, a peptide containing an extra Asp in the TM region might have a higher pK a and might be directed to tumors more effectively. Another important factor to be considered is the broadness of the pH transition of the peptide, which is dictated by the cooperativity of the transition. On one hand, for the case where the peptide pK a is lower than the tumor pH e but the transition is broad (m value is low), a significant part of the pH transition could intercept the pH e value, resulting in a significant pHLIP tumor insertion. However, such a scenario will also lead to more accumulation in healthy tissue. Since it is usually desirable to have a high tumor/organ ratio, an insertion transition of high cooperativity might be best. This would ensure greater differentiation between the amount of inserted peptides and the amount of noninserted peptides over a narrow range of pH values, favoring selective tumor targeting, since the difference in pH between normal tissue and cancerous tissue may be only 0.5-0.7 units. However, we must bear in mind that the measured pH e provides an indication of the average acidity outside the cell for a given tumor and can vary between different tumor regions. Furthermore, pH e may not reflect the precise pH on the exterior surface of the cells, since the cells pump protons to the extracellular medium and ΔpH will lead to proton accumulation at the membrane surface. 25 Another feature that is expected to shift the equilibrium toward the membrane-inserted form is the presence of Asp/Glu residues at the C-terminus of the peptide. After being translocated across the plasma membrane into the cytoplasm, where the pH is neutral, these groups would be deprotonated. Since the translocation of charges across membranes is unfavorable, the inserted form would be stabilized.
pHLIP shows promise as a means of targeting cells in acidic tissues and delivering agents for therapy and imaging. At the same time, we are learning more about the binding and insertion of peptides at the membrane surface. Here we have shown that variation in the positions and numbers of carboxyl group titrations modulates the pK and cooperativity of insertion.

Peptide synthesis and assessment of monomeric state
Peptides were made by solid-phase synthesis, using standard 9- The peptides described in Table 1 were used in the experiments, except for some experiments with D2-D0, where a Cys-less version was employed (similar results were obtained for both results; data not shown).

Analytical ultracentrifugation
Sedimentation velocity experiments were performed at 25 °C in a Beckman Optima XL-I analytical centrifuge at 35,000 rpm. Peptides at a concentration of 7 μM were dissolved in 5 mM phosphate buffer (pH 8) after 1 h of incubation at room temperature. Absorbance at 280 nm was used to monitor centrifugation, and analysis was performed using SEDFIT. 26

Liposome preparation
The required amount of chloroform-dissolved POPC (Avanti Polar Lipids) was placed in a glass tube, dried with argon, and then held under vacuum overnight. The dried film was resuspended in water or 10 mM phosphate buffer (pH 8) and vortexed.
Extrusion to make unilamellar vesicles was performed using a Mini-Extruder (  For determination of spectral maxima, we used the FCAT mode of the PFAST software, which fits the experimental spectra to log-normal components. 27 Equation (1) where F a = (f A + S A pH) and F b = (f B + S B pH); f A and f B are the spectral maxima for the acidic and basic forms, respectively; S A and S B are the slopes of the acidic and basic baselines, respectively; and m is the cooperativity parameter. Fitting by nonlinear least squares analysis was carried out with Origin software.

Circular dichroism
Samples were prepared as in the fluorescence experiments, but the final molar lipid/peptide ratio was 300:1, with the final peptide concentration varying from 2 to 5 μM. CD spectra were recorded in Jasco J-810 and MOS450 Biologic spectropolarimeters interfaced with a Peltier system. Spectra were recorded at 25 °C using 2-or 5-mm cuvettes, the scan rate was 50 nm/min, and 10-30 averaging steps were performed. Raw data were converted into mean residue ellipticity according to 30 : where Θ is the measured ellipticity, l is the path length of the cell, c is the protein concentration, and N is the number of amino acids.
For the study of membrane attachment, insertion, and its reversibility, the typical procedure was as follows: The samples were incubated with POPC vesicles at pH 8 for 90 min, the spectra were recorded, the pH was lowered to 4.0, and the measurements were performed after 30 min. Finally, the pH of the sample was

OCD measurements
For OCD measurements, supported bilayers were prepared on quartz slides with 0.2mm-thick spacers on one side and with a special polish for far-UV measurements

Biotin translocation assay
HABA dye (4′-hydroxyazobenzene-2-carboxylic acid) binds to avidin at a 1:1 stoichiometry and absorbs at 510 nm only in the avidin-bound state. This interaction is strongly displaced by the binding of biotin to avidin, resulting in a quantitative reduction in HABA absorbance. This property was used to probe the location of the Cterminus of different peptides with regard to the liposome (inside or outside) (method modified from Nicol et al. 31 ). The C-terminus of each of the peptide variants was labeled with biotin (see the text below). The rationale for the assay is that pH-driven biotin at its C-terminus was taken as 100% reversibility, and that of pHLIP labeled at its N-terminus was taken as 0%.
Peptides were labeled at the C-terminal Cys residues using the membrane-  Tables   Table 1. Sequence of the peptides. 31 33 a The pHLIP sequence is referred to as wt.
b The variant peptides are named by a D followed by the number of aspartic acid residues in the TM and C-terminal regions. Two different D3 peptides were studied, D3a and D3b, each with different transmembrane aspartic acid residues mutated. The acidic residues that are expected to interact with the hydrophobic core of the membrane at some stage of the insertion process (Asp 14, 25, 31 and 33, in red) were mutated to the polar residues marked in bold. The N-terminal Asp-tag and the Leu26Gly mutation are highlighted in italics. The transmembrane region of pHLIP was predicted, using the octanol scale 5 , to be located between residues Ile7 and Leu29 (marked with inverted blue triangles). N-and C-terminus were not capped.           There are no conflicts of interest.

Introduction
The rapid growth and division of tumor cells creates an enhanced need for glucose and other nutrients, which the cells take up at a high rate, overwhelming their mitochondrial capacity to use all of the glucose efficiently (1). The result is aerobic glycolysis, which elevates lactate and proton production: the "Warburg" effect (1, 2).
Further, some tumors are starved for oxygen, resulting in even more glycolytic acid production (3,4). Under the resulting low pH conditions, normal cells have a tendency to undergo p53-induced apoptosis (5, 6), whereas cancerous cells invoke alternative routes, manipulating ion fluxes with proton extruders and other transporters to afford continuous survival (7). Pumping the acidic components out of the cell maintains cytoplasmic pH and enhances the pH gradient (ΔpH) and the cellular exterior surfaces become more acidic than those of cells in normal tissues (8). The level of extracellular acidification, however, is variable, depending on (i) the reliance of the malignancy on glycolysis (9-12), a phenomenon resulting from the pleiotropic adaptation of cancer cells towards a glycolytic phenotype, (ii) the impact of variation in the distal vascular delivery of nutrients, and (iii) the state of hypoxia (13,14). The low pH environment stimulates cell invasion, angiogenesis and finally, metastasis (15)(16)(17).
Tumor acidosis could be a useful biomarker for selective drug delivery, targeting and delineation of malignancies. With the discovery of a membrane-inserting peptide (pHLIP) that preferentially binds to cell membranes at low pH, practical clinical imaging and delivery of therapeutic payloads may be possible (18)(19)(20)(21)(22). At normal pH, pHLIP binds as a largely unstructured peptide at a membrane surface, but at acidic pH it folds and inserts across the plasma membrane as an alpha helix (23).
We have previously demonstrated that pHLIP might be useful as a PET (Positron Emission Tomography) probe with 64 Cu (t 1/2~1 2.7 h) (24). Tumor uptake in prostate cancer models was achieved, and related to a low extracellular pH (pHe), but shortcomings were apparent (24). The success of the probe as a marker of acidosis was found to have contrast and clearance complexities associated with the pharmacokinetics (PK) of pHLIP, warranting further development efforts. Targeting of fluorescent pHLIP variants were recently studied, and a range of potential properties was found, including altered kinetics of insertion (Scheme 1) (25). Here, we describe a much improved PET probe that was developed using three strategies:

Results
Appropriate peptide sequence, radionuclide and chelating ligand modifications can significantly improve pHLIP-PET properties.  Table 1). Binding assays using PC3-wt prostate cancer cells in different pH-buffered environments (pH~ 6.3, 6.7, 7.0) showed that these peptides target cells at low pH. The binding activity of each radiolabeled pHLIP variant, expressed as "% Bound" normalized to the added amount of probe, is displayed in Fig. 1. Variants WT and Var7 were selected as lead compounds for small animal PET imaging and biodistribution studies in vivo, due to their differential but favorable binding at low pH and significantly lower uptake at neutral pH, resulting in an improved dynamic range/contrast in the pH range of interest (pH 6 -7.4). The control peptide, K-WT, showed a reverse trend, with enhanced binding at high pH.

In Vitro
The lysine residues in K-WT are in their charged form at low pH, inhibiting membrane insertion, while in a more alkaline environment these lysine residues may be partially protonated, enhancing peptide-membrane interaction (29).  Table 1 . 2).
A direct comparative analysis between the two 68 Ga-labeled probes demonstrated a higher tumor uptake with the shorter sequence compared to the parent WT. The blood residence at 4 h p.i. was similar; however, slightly increased nonspecific tissue binding was demonstrated by 68 Ga-DOTA-Var7 ( Fig. 2A). Compared to the WT peptide, the kidney uptake for Var7 was elevated, which can be rationalized as resulting from faster probe clearance. Comparing tumor-to-background ratios of both radiotracers in Table 2 2B). The blood residence activity improved with a final tumor-to-blood ratio of 2.63 ± 0.57 at 24 h p.i. (Table 3). Despite improvements made on the pHLIP backbone, concerns still remained with radiotracer retention in key organs. Hepatic uptake of the radiotracer displayed unremarkable retention over 24 h with 6.05 ± 1.36 %ID/g ( Fig.   2B-C), similar to the values reported for the 64 Cu-DOTA-WT construct (4.88 ± 0.98 %ID/g at 24 h) (24); this uptake is likely to be from random scavenging of radioactive metabolites, including de-metallated 64 Cu in the liver (30,31). The tracer distribution in the kidney revealed only nominal reduction, even after 24 h (19.6 ± 4.0 %ID/g), likely due to the renal acidic environment (pH~5), which is expected to cause binding of these pHLIP variants for a period of time (32), but possibly including other effects, since it could be improved (see below).
Our efforts to limit indiscriminate tissue accretion of pHLIP PET probes led us to seek improvements of the radiometal-chelate stability and the resistance to proteolytic degradation. Var7 was modified with the NOTA ligand. In addition, since previous reports described superior chelate affinity for 64 Cu (33)(34)(35) and, in addition, we employed D-amino acids (named Var7(D) from now on), known for resistance to enzymatic proteolysis compared to L-peptidomimetics (36)(37)(38). Similar 64 Cu radiolabeling conditions were employed as described above. In PC3-wt xenografts, no differences in tumor accretion were seen between 64 Cu-NOTA-Var7(D) (Fig. 2B, SI Table 4) and the DOTA scaffold (Fig. 2B, SI Table 3).
The pH-dependent changes in circular dichroism (Fig. 3A) and tryptophan fluorescence signals (Fig. 3B) are similar to those observed for the peptide with no chelate and metal (25) indicating pH-dependent interaction of the pHLIP portion with the membrane. The apparent pKa of insertion was ~ 5.9 (Fig. 3C), which is slightly higher than for the peptide alone (5.5), probably due to the presence of the chelate.
The log P value of 64 Cu-NOTA-Var7(D) was measured as -2.45 ± 0.13, revealing a significantly polar compound. The properties of increased solubility and the elevation of the pK of insertion may contribute to its improved properties in vivo.

Probe accumulation correlates with acidity.
We wanted to explore disparities, if any, in the extacellular pH (pHe) of tumors with and without pHe regulators, particularly in tumors transduced to overexpress CAIX, a carbonic anhydrase elevated in tumor cells to cope with high CO 2 production.  Fig. 4B).
Uptake of 64 Cu-NOTA-Var7(D) correlates inversely with pHe when data from all three tumor models are taken into account. Each mouse used for pH MRS measurements was also used for PET and biodistribution experiments, giving greater confidence in the correlations (for pairing details see SI Table 6). In the plot of pHe versus 64 Cu-NOTA-Var7(D) uptake (PET imaging at 1 h p.i. and 24 h ex vivo tissue sampling radioactivity assays) taken from the distribution studies (Fig. 5A), incremental accumulation of the radiotracer is seen as the tumor acidity increases. By pooling all data points from all prostate xenografts (Fig. 5B), threshold limits can be established from the data, showing that a tumor pHe < 6.9 provides high probe localization (> 3.0 %ID/g), whereas a pHe range of 6.9 -7.4 results in lower probe uptake (< 3.0 %ID/g).

Ex vivo autoradiography demonstrates pHLIP accumulation in tumor regions associated with elevated metabolism
Histological staining was used to examine viability and metabolic features of the tissues that stain or do not stain with the probe. uptake. There appeared to be no relationship between Hoechst 33342 staining intensity and 64 Cu-NOTA-Var7(D) uptake. Thus, we find that probe uptake is correlated with hypoxia and LDH-A.

Discussion
By creating a useful probe for imaging tumor acidosis, we enable assessment of a universal trait associated with tumor invasiveness in most malignancies. We illustrated the improvements made toward better PK and dosimetric properties of pHLIP as a non-invasive PET radiotracer. More importantly, this probe was able to distinguish highly acidic tumors, with a direct association to tumor pHe. Furthermore, we extended our efforts to understanding the mechanism of uptake of this probe through autoradiographic and histologic studies of all three tumor models to provide insights on its target. Our results differ in some respects from those reported earlier. In our hands, comparison of two of the tumor models (LNCaP and PC3 wt) in the right shoulder of athymic nude mice, the pHe showed a trend opposite to that observed by Vavere et al. (24). They also used LNCaP and PC3 but for tumors implanted in the flanks of athymic nu/nu mice and for tumor volumes > 500 mm 3 , so the observed differences may potentially be due to the smaller tumor size (< 400 mm 3 ) used in our study and the differences in tumor location (shoulder vs flank). Our goal was to use tumors with only moderate necrosis for best comparison with tumors seen in the clinic, hence our choice of small to medium-sized tumors. Further, we used 1 H decoupled 31 P MRS, which may influence the average chemical shift of 3-APP, since without 1 H decoupling the signal shape and width is not only determined by T 2 relaxation and the pHe tissue distribution, but also by the multiplet structure of 3-APP (43). We did not find a significant relationship between tumor size and pHe for tumors < 400 mm 3 (SI In retrospect, we find that the outcomes of measuring pH as an average do not give a true representation of tumor acidity, as evidenced by the broad pH distributions observed from 31 P MRS. Instead, details of pH variation within a tumor may be key, even at the cellular level. Variations in the spatial distribution of pHe have been reported such that gradients exist at the interface of the cellular membrane and cytosol (45,46), prompting us to examine the differences between cytosolic and extracellular pH, and to derive the net proton flux (although we still needed to use average values).
We observed that the transduced PC3-CAIX and the LNCaP implants had greater extracellular pH gradients (∆pH) than the wild type (PC3-wt) model; however, the measured pHe values of the two models followed an opposite trend from the ∆pH values. We rationalize that these contrasting measurements may be due to the vast heterogeneity in tumor homeostasis and development, governed by an intricate mesh of metabolic pathways including rate of glycolytic metabolism, expression of acid extruding protein, and diverse buffering capacities and O 2 concentrations in the blood vessel network, to name a few (44,47,48). Despite these uncertainties, we observed a correlation of targeting with absolute pHe, where at a pHe < 6.9, higher tumor accumulation of the radiotracer was observed, with > 3 %ID/g. However, at pHe > 6.9, measuring and imaging tumor acidity using this probe is poorly resolved. We postulate that this may be an effect of the insertion pKa of the full construct (pKa ~ 5.9).
The  Fig. 5) that display high uptake of the hypoxia marker pimonidazole, but shows no clear relationship to the vascular perfusion marker Hoechst 33342 (Fig. 6). The LDH-A-mediated conversion of pyruvate to lactate is postulated to be one of the principal sources of tumor acidity (49,50); elevated LDH-A would be expected to result in concomitant elevated pHLIP binding.
LDH-A expression, while previously been shown to be hypoxia-regulated (via the HIF-1 transcription factor), has not yet been individually validated as a marker of low pHe (51). However, for these studies we took elevated expression of LDH-A as a stable marker of regional lactic acidosis, which is not susceptible to perfusionmediated fluctuations in extracellular microenvironment (52). The predicted cellular half-life of LDH-A is tissue-type dependent, but is generally in the order of several days (53), which is appropriate to our experimental protocols.  (10,46,48). It is likely that tumor regions of poor vascularity and low pO 2 will also possess excess extracellular H + ions due to anaerobic glucose metabolism and local lactic acidosis. While we observed a trend towards increasing 64 Cu-NOTA-Var7(D) uptake with increasing pimonidazole uptake, the relationship appears to be non-linear (Fig. 6C-D). This may in part be due to the effect of pHe on absolute pimonidazole uptake, although this is likely to be a minor effect over the pHe ranges measured in this study (56).
By finding a probe that marks the acidosis inherent in tumor metabolism, we have  corrected, and the α-NDP/α-NTP signal calibrated to -10.05 ppm (Fig. 4A). The MR spectra were fitted in the time domain, using the software package XsOsNMR (kindly provided by Dr. Dikoma Shungu and Xiaoling Mao) and the intracellular and extracellular pH (pHi, pHe) calculated from the chemical shifts of inorganic phosphate (Pi) and 3-aminopropylphosphonate (3-APP) respectively, described previously (43).
The inorganic phosphate signal, Pi represents primarily intracellular pH (pHi) (58, 59). phosphor imager. Following autoradiographic exposure, the same or sequential sections were then used for fluorescence and H&E staining and microscopy.
Immunofluorescence staining for pimonidazole was carried out as previously described (54), the major difference being the use of a rabbit polyclonal antipimonidazole primary antibody (NPI). Secondary detection was carried out using goat anti-rabbit Alexa-488 (Invitrogen, Grand Island, NY) (1:100 in blocking buffer).

Pixel re-binning and scatterplot generation
Pixel re-binning was done using an adaptation of the methods described in (61) and (62). Briefly, registered image sets were re-sampled to 50×50×10 μm 3 pixel size, each image converted to an 8-bit grayscale image, and pixel values with their corresponding image location recorded. The data from the DAR image was designated as independent and the fluorescence image data as dependent. Data were then sorted in ascending order of the independent variable while maintaining the association between independent and dependent values. The data set was then split into deciles, each containing the same number of data points, i.e. the 10% of the data points lowest in terms of the independent variable, then the next lowest 10%, etc.

Statistical Analysis
Data values were expressed as the mean ± S.D. unless otherwise stated. Statistical analysis was performed using GraphPad Prism version 5.03 software using student's ttest. A P value of < 0.05 is considered statistically significant.           Immune complexes were detected by horseradish-peroxidase labeled antibodies and enhanced chemiluminescence reagent (Amersham, Buckinghamshire, UK).

MRS
The MR experiments were performed on mice using a horizontal-bore 7T MR  (Fig. 4A). The MR spectra were fitted in the time domain, using the software package XsOsNMR (kindly provided by Dr. Dikoma Shungu and Xiaoling Mao) and the intracellular and extracellular pH (pHi, pHe) calculated from the chemical shifts of inorganic phosphate (Pi) and 3aminopropylphosphonate (3-APP) respectively, as described in detail previously (3).
As the inorganic phosphate signal in tumors is predominantly comprised of intracellular Pi, due to the densely-packed cells, the pH calculated from its chemical shift represents primarily intracellular pH (pHi) (4,5). pH values were reported as the mean ± standard error of the mean (SEM). Table 1. Tissue uptake (mean %ID/g ± S.D.) of 68 Ga-DOTA-WT administered via lateral tail vein in male, athymic nu/nu mice bearing PC3-wt prostate cancer xenografts.  Table 5. The apparent pK (pKa) of pHLIP peptide insertion into membrane, the sedimentation coefficients (Sed. Coeff.) and calculated molecular masses of the peptides in solution at pH 8.0, and the spectral parameters of peptides in the states I, II and III are presented. The spectral parameters were obtained from the analysis of the fluorescence and CD spectra: the maximum position of the fluorescence spectrum  m , Sthe normalized area under the spectra (normalized with respect to the area under the spectrum of pHLIP in state I);  225 x 10 3 , deg cm 2 dmol -1the molar ellipticity at 225 nm. 340/3.38/-7.23 * The pKa was not calculated for WT, since there is no shift of the position of maximum of fluorescence spectra between states I, II and III. Table 6. Table detailing the values obtained from the independent in vivo MR, in vivo PET, and ex vivo experiments performed and the corresponding pairing as used in the figures. Tumors used for autoradiography and immunohistochemistry (green highlight) were not used for biodistribution studies. Abbreviations: BL pHiintracellular pH before 3-APP injection, pHiintracellular pH after 3-APP injection, pHeextracellular pH determined from 3-APP signal, ΔpHi = BL pHi-pHi, ΔpH = pHi-pHe, Ttumor, T/Mtumor to muscle ratio. Data in red depict experiments with missing values, and were not included in the analyses.  Fig. 1 Serial PET images of a representative PC3-wt prostate tumor obtained after 1 h, 2 h and 4 h post injection of 68 Ga-DOTA-WT, demonstrating non-specific binding of the pHLIP variant to healthy tissue (e.g. liver, kidney). The white circle delineates the subcutaneous shoulder tumor.  Table 5. Hydrophobic mismatch results in significant energetic penalties, which can lead to structural perturbations in a polypeptide, alteration in a polypeptide's mobility and/or membrane thickness changes (5-7).

SI
Here we have performed a comparative investigation of long and short pHLIPs (pH Low Insertion Peptides) interactions with the lipid bilayer of a membrane. Long-pHLIPs are well investigated water-soluble membrane polypeptides, which insert into a lipid bilayer and form a stable TM alpha-helix as a result of a drop in pH (8)(9)(10)(11). The insertion of the peptides of pHLIP family is employed for the targeting of acidic diseased tissue including tumors and intracellular delivery of polar cell-impermeable cargo molecules across membrane (8,(12)(13)(14)(15).
Truncated versions of pHLIPs (short-pHLIP), if inserted into membrane, should create negative hydrophobic mismatch. We used several spectroscopic assays to prove that short-pHLIP's interaction with a lipid bilayer at low pH leads to the membrane inserted state of the peptide. Small angle x-ray scattering (SAXS) experiments performed on long and short peptides allowed us to demonstrate the thinning of a lipid bilayer of membrane as a result of short-pHLIP's insertion into the bilayer. The experimental design was based on the comparison between interactions with membranes of well-characterized long-pHLIPs and truncated short-pHLIP.

Methods and Materials
The detailed information about all methods could be found in Supporting Information.
All liposomes (labeled at inner leaflet) were incubated with the peptides and FRET from tryptophan residues to NBD at inner leaflet of the bilayer was monitored.
Synchrotron SAXS measurements were carried out at beamline ID02 of the ESRF in Grenoble, France (16). The measured two dimensional SAXS patterns were normalized to an absolute intensity scale using the standard procedure and azimuthally averaged to obtain the intensity profile as a function of q. The background buffer was also measured and subtracted from each averaged sample intensity profile before fitting the data.

Results
The main focus of our research is an investigation of interactions with lipid bilayer of membrane of short-pHLIP peptide, which is a truncated version of full-length pHLIPs (long-pHLIPs). Our experimental strategy is a comparison between membrane interactions of short-pHLIP and well-characterized long-pHLIPs.

Short-pHLIP: AEQNPIYWARYADLLFPTTLAW
Long-pHLIP: AEQNPIYWARYADWLFTTPLLLLDLALLVDADET Long-pHLIP*: AEDQNPYWRAYADLFTPLTLLDLLALWDG In the dual quenching and FRET spectroscopic assays we used long-pHLIP* peptide (with truncated flanking sequence), which has Trp residues located at the beginning and end of TM part, as in a short-pHLIP. We demonstrated previously truncated long pHLIP adopts TM helical orientation in membrane at low pH similar to a full-length long-pHLIP (11). We also attempted to investigate single-Trp mutants of short and long pHLIPs with the goal of simplifying the interpretation of spectroscopic data.
However we found that some single-Trp mutants of short peptide did not exhibit pHdependent properties and most probably do not insert into membrane (further investigation is needed). Therefore, here we present results obtained with long and short pHLIPs containing both tryptophan residues.
Previously we demonstrated that long-pHLIPs insert into the lipid bilayer of To establish location of tryptophan residues (thus pHLIPs) within a lipid bilayer of membrane a dual quenching assay was employed (18). Effective quenching of Trp fluorescence by acrylamide occurs when tryptophan residues are exposed to polar parts of the outer or inner leaflets of a bilayer. At the same time tryptophan residues located in the middle of a membrane could be effectively quenched by another quencher of tryptophan fluorescence, 10DN. We performed dual-quenching assay at pH 8.0 and pH 4.0 for short and long pHLIPs both containing Trp residues at the beginning and end of the expected TM region of the peptides (Figure 2). At pH 8.0 short-pHLIP just barely partitions into the bilayer and therefore tryptophan fluorescence is quenched by acrylamide very well (Figure 2a and Table 1). Long-pHLIP* being more hydrophobic, is located much deeper into the bilayer, which correlates well with our previous data (11,19). Lowering the pH reduces quenching of Trp fluorescence by acrylamide (from 79.1 to 48.1% for short-pHLIP and from 44.1 to 31.1% for long-pHLIP) and increases quenching by 10DN (from 12.3 to 34.7% for short-pHLIP and from 32.7 to 44.6% for long-pHLIP) (see Figure 2 and Table 1). The overall trend of short-pHLIP's partition into bilayer at low pH is similar to long-pHLIP's. However, Trp residues in short-pHLIP are more exposed to acrylamide compared to Trp residues of long-pHLIP. According to our published data the truncated peptides have a lower affinity to the bilayer compared to long pHLIPs (8), thus higher amount of short-peptides could be found in solution, which will lead to the enhanced quenching by acrylamide.
The dual-quenching assay provides information about degree of partitioning of Trp residues into bilayer. However it does not allow distinguishing between the inner or outer leaflets location of acrylamide-accessible Trp residues. To further investigate location of tryptophan residues in membrane we also performed NBD-FRET assay (20,21). First, symmetrically-labeled (with NBD dye) POPC liposomes were prepared. Then, membrane-impermeable dithionite was used to chemically modify and quench the fluorescence of NBD in outer leaflet of the bilayer, followed by the removal of dithionite by gel filtration. As a result, asymmetrically-labeled liposomes with NBD at the inner leaflet were obtained. The absence of potential flip-flopping of lipids was accessed by absence of quenching of NBD fluorescence by addition of dithionite. FRET was monitored from the tryptophan residues of peptides to NBD.
Energy transfer occurs when both fluorophores (Trp and NBD) are in a close proximity to each other (the Förster distance for Trp-NBD donor-acceptor pair is about 10 Å (22)). Thus, when tryptophan residues are located in the outer leaflet of the bilayer, there is no any significant energy transfer to NBD at the inner leaflet. This is the situation that was observed at pH 8.0 for both peptides, but was less pronounced for long-pHLIP, which partitions deeper into the membrane. At the same time, at low pH the FRET signal was comparable for both peptides (Figure 3b, d, red lines). We observed that the NBD fluorescence signal increased by 11.7 and 12.9 times for short-pHLIP and long-pHLIP, respectively, in the presence of POPC at low pH compared to the baseline. It indicates that Trp residues (located at the C-terminus) in long and short pHLIPs both are in close proximity to the headgroups of inner leaflet of bilayer. Thus, we can conclude that short-pHLIP inserts into lipid bilayer of membrane and spans bilayer similar to long-pHLIP.
When a short peptide is inserted into a membrane, as it is well known, a hydrophobic mismatch is created. We already mentioned about the possibility of presence of some elements of 3 10  can indicate on insertion of short-pHLIP into bilayer. We can conclude that membrane thinning occurs to reduce a hydrophobic mismatch and an overall energy of the system.

Analysis of SAXS data
The scattered intensity can be expressed as I(q)F(q) 2  (23), where F(q) is the size averaged scattering form factor of vesicles, which is the Fourier transform of the bilayer electron density profile. By describing the electron density (ED) profile along the normal of the phospholipid bilayer of outer leaflet headgroups, hydrocarbon tails and inner leaflet headgroups as a sum of Gaussian functions (23)(24)(25)(26)(27): we can obtain the following expression for the scattered intensity: where  k , z k and  k are the relative weight, position and width of the k-th Gauss function, respectively. For the bilayer k = 1 represents inner headgroups, k = 2 represents hydrocarbon tails, and k = 3 represents outer headgroups. We assume that the center of the bilayer coincides with the center of the hydrocarbon tail, which means z 2 will be close to zero. The ED of liposomes in absence and presence of peptides was fitted by the sum of exponential functions. Fitting was performed using Origin 9.0. The best fit was defined as the one with the smallest  2 .

Discussion
Previously we established that at the low lipid:peptide ratios we used in this study, the peptide is adsorbed to 50-60 lipids on average and an additional 50-60 lipids are perturbed. In contrast, a peptide in the transmembrane state III is estimated to affect only ~22 lipids, roughly one layer around the helix (10). Also we showed that long-  (19). The results of SAXS measurements indicate a 12% and 7% thinning of the lipid bilayer when long-pHLIP or short-pHLIP occupy the outer leaflet of bilayer at high pH, respectively, compared to the same liposomes with no peptide. Our results are also in agreement with the data obtained by Huang and co-authors indicating that amphipathic peptides, which adopt helical conformation at the surface of bilayer, induce membrane thinning (29,30). When a peptide is adsorbed into the surface of a bilayer, it pushes the lipid headgroups aside. Since the total volume of the chains is constant, this causes the membrane to thin. Thus, polypeptide that is adsorbed by a bilayer even in coil conformations, like in the case of pHLIPs, induces some membrane tension and stress, which leads to the membrane thinning. The interaction of short pHLIP with a membrane causes the membrane thinning to a lesser extent, since the affinity of truncated pHLIPs to lipid bilayer at pH 8.0 is lower (8), However, there is no disallowed region between the alpha-helical and the 3 10 -helical conformations in the Ramachandran plot, and therefore transitions between helices can easily occur (44). Furthermore, the hydrophobic environment of protein interiors or lipid membranes could stabilize the 3 10 -helix (45). There is a high probability to observe 3 10 -helical segments as N-and C-terminal capping of an alpha-helix. The mixture of helical structures in a membrane and their transition from one to another was demonstrated to be important for biological function (31)(32)(33)(34). Our data does not point to the aggregation of the peptide in membrane; however we cannot exclude that as a possibility. We can confirm the thinning of a bilayer as measured in SAXS experiments and the formation of a stretched 3 10 helix or most probably mixture of alpha-and 3 10 -helices with 3 10 components at the beginning and end of alpha-helix.

Author Contributions
Tables

Introduction
Acidity is associated with development of various pathological states such as tumors, ischemic stroke, neurotrauma, epileptic seizure, inflammation, infection, wounds and others(1-3). Thus, it becomes increasingly important to be able to measure pH with accuracy, precision, and high spatiotemporal resolution in experimental systems of cell culture, animal models and in human beings.
The pH electrodes are used for accurate pH measurements in solution. As such microelectrodes were the first method used to probe pH in living tissue. However they are highly destructive to the tissue and are weighted to the extracellular pH (4,5). Later on noninvasive pH measurement methods were developed that could measure either the pHi , intracellular pH, pHe, extracellular pH, or both. PET has been used for measuring tissue pH using radiolabeled dimethadione, which distributes in intracellular and extracellular space according to the pH gradient across membranes (6). Unfortunately, dimethadione's distribution depends on the transmembrane pH gradient and the fractional volumes of intra-and extracellular space, both of which are unknown. In vivo MRS and MRI have been used to monitor metabolic and physiologic processes employing endogenous and exogenous nuclear MR-active compounds (7).
MRS methods are generally based on a difference in chemical shifts between pHdependent and -independent resonances. Several isotopes have been evaluated to determine tissue pH with MRS. 31 P-MRS provides a robust technique for simultaneously measuring pHi from the chemical shift of endogenous inorganic phosphate and pHe from the chemical shift of exogenous indicators, such as 3-aminopropyl phosphonate (8). Tumor pH was also measured using hyperpolarized 13 C bicarbonate (9, 10). One of the limitations of dynamic nuclear hyperpolarization is that the hyperpolarized nuclear spin signal decreases rapidly according to spin-lattice relaxation, T1. Therefore, measurements must be completed within 1-2 min after injection. Another approach using MRI relies on perturbing the relaxivity of water via pH-dependent relaxation agents such as tetraphosphonate, gadolinium-DOTA-4AmP52 (7, 11).
The described above studies showed that tumor pHe is heterogeneous and acidic.
However, these methods are still limited in spatial resolution and cannot measure pH on a cellular level. Only optical methods can provide cellular resolution. Fluorescence imaging was employed to study pH at the surface of cultured cancer cells and monitor behavior of individual fluorescent cancer cells in the heterogeneous microenvironments of tumors (12)(13)(14)(15)(16). For pH-imaging mostly pH-sensitive dyes, fluorescence intensity of which is changed in a response of pH, are used. However, accurate calibration for the probe concentration is needed. pH-sensitive and pHinsensitive fluorophores were used to functionalize the bacteriophage particles with many copies of these dyes and perform in vivo imaging (15). However, the bacteriophages particles are taken by endocytosis, thus reporting pH primarily in endosomes. Fluorescence lifetime imaging is based on measurements of a fluorophore's excited state lifetime, which changes in accordance with pH alterations.
However, lifetime measurements are more complicated, especially for the measurements in the nano sec range, which is a typical lifetime of most of organic dyes (including pH-sensitive ones). One of the approaches is to use long-lived (micro seconds) metal-chelate complexes (they mostly exhibit phosphorescence signal).
However, most of them have short wavelength of excitation (<450 nm), which has low tissue penetration (17,18). Despite the fact that optical imaging can provide singlecell resolution in vivo, in order to measure pH on the surface of individual cell the pHsensitive probe needs to be located close to the plasma membrane. In most cases, the pH-sensitive agents were small molecules distributed in entire organ/tissue and blood (where pH is normal) and washed out from the body very quickly. In case of use of nanocarries (nanoparticles or bacteriophage particles), cells internalize them readily via endocytotic pathway, thus pH could be reported predominately in endosomes. The use of antibody or receptor targeting peptides/molecules could also lead to their internalization. The lipids or fatty acids conjugated with pH-sensitive probes (13,19) could be used for pH measurements on cellular level, but they are not selective to cells in disease site, they will incorporate into any cellular membrane, and can readily undergo flipping and participate in lipid exchange, thus making problematic identification of their exact location, especially in in vivo experiments.
We propose a novel approach of pH measurements at the surface of cells, which is based on use of a pH-sensitive fluorescent dye SNARF conjugated to a pH Low Insertion Peptide (pHLIP). Peptides of pHLIP family insert into the lipid bilayer of a membrane in a pH-dependent manner exposing N-terminus to the extracellular space and translocating C-terminus across membrane into cytoplasm (20)(21)(22)(23). The molecular mechanism of pHLIP action is based on protonation of Asp/Glu residues, which enhance peptide hydrophobicity and promotes membrane-associate folding and formation of transmembrane helix (24,25). pHLIP labeled with optical, PET or SPECT probes target acidic diseased tissue and are considered to be novel acidity markers (26)(27)(28)(29)(30)(31)(32)(33). A novel tool for mapping pH at the extracellular surface of cell, we introduced here, might open an opportunity to contribute in understanding of diseases progression and development of approaches of pH-based image-guided interventions.

Peptide Synthesis and Conjugation with Fluorescent Dyes
A pHLIP peptide with a single Lys residue at the N-terminus (the N-terminus is  with a diffraction grating of 300 l/mm blaze 500 nm, 400 µm entrance slit and a Newton EM EMCCD (Andor) camera thermoelectrically cooled to -60˚C. Spectra were taken every several minutes until 3 in a row were identical. After spectra recording, the fluorescent images were acquired using Qcapture software by a Retiga-SRV CCD (Qimaging) with two emission filters FF01-580/14-25 (Semrock) and FF01-640/14-25 (Semrock) with transmittance at 580 ± 10 nm and 640 ± 10 nm, respectively.

Trypan Blue Assay
Trypan blue solution (Sigma) at concentration of 0.67 M in experimental PBS of pH 7.0 was added to a HeLa spheroid, which was incubated with SNARF-pHLIP as described above, in a glass bottom collagen coated cell dish (MatTek). The fluorescence spectra and images before and immediately after addition of Trypan Blue were taken as described above with 20x objective.

In vivo Imaging of Tumors
When tumor reached 5-8 mm in diameter, mouse was subjected to a starvation (no food) for 24 hours before a single tail vein injection of 4 nmol (100 μL of 40 μM) of SNARF-pHLIP in PBS was performed. At 4 hours after construct administration, the skin was removed from tumor site under ketamine/xylazine anesthesia and mouse tumor was placed onto a 24 x 60 mm NO 1 thickness glass slide and imaged on fluorescent microscope using objective with 20x magnification as described above.
The SNARF-pHLIP fluorescent spectra and images were taken from various areas of tumor before and after an intra-peritoneal injection of 125 mg of glucose (125 mg in 220 µL of PBS pH 7.4). After in vivo imaging animal was euthanized by cervical dislocation and tumor was excised, cut in half and fluorescence spectra and images were acquired immediately as described above.

Ex vivo Imaging of Tumors
When

Spectra and Image Analysis
The fluorescent spectra and images of SNARF were analyzed by our programs. The spectra were analyzed with a Mathematica program (Version 10, Wolfram), and images were analyzed using Matlab program (Mathworks) both introduced by us. The description of the program is provided in the Result section. All graphs were constructed using Origin Lab (Version 9.1, Origin Lab Corporation). The p-level values were computed based on the two-tailed test. is a highly metastatic, while NM2C5 is non-metastatic (34,35).

Various
Our approach to measure pH at the surface of cells is based on use of WT-pHLIP, which inserts into a cellular membrane and forms a transmembrane helix translocating the C-terminal end into the cytoplasm and exposing the N-terminal end to the extracellular space. Additionally the pHLIP has multiple protonatable residuesat the membrane-inserting C-terminal end, which are deprotonated in the cytoplasm and serve as additional anchoring point for the peptide in membrane. Thus, once WT-pHLIP is inserted into a plasma membrane, the rate of its exit from membrane is very low even when extracellular pH is raised. This opens up an opportunity to treat cells with WT-pHLIP at low pH and then raise pH of media for measurments. The acetylated N-terminus of the peptide contains a single Lys residue, which was conjugated with a SNARF-1 dye and this dye was subsequently converted to its fluorescent form by chemical activation. The product was purified and characterized and used in all experiments.
Our choice of ratiometric pH-indicator, SNARF, was dictated by the fact that pH values could be established independent of the dye's concentration, which was used previously to measure pH in vivo (16). SNARF also has other desirable characteristics such as high excitation and emission wavelengths, two fairly fluorescent peaks and it runs in a single excitation dual emission configuration. The SNARF-pHLIP was excited by the xenon lamp attached to the inverted epi-fluorescent microscope in the range of 531 ± 20 nm as selected by an excitation filter. The emission was detected by two different set ups: i) fluorescence was passed via emission cut off filter for the detection of light at wavelengths from 580 nm and higher. The spectrograph connected to the microscope allowed to record entire fluorescence spectra from 500 to 800 nm simultaneously ( Figure 1A). Our program in Mathematica performs a smoothing of spectra and establishes the emission maximums of the SNARF-pHLIP and their ratio The main idea of our approach is to measure pH at the surface of cells. To prove that SNARF is indeed located in the extracellular space we imaged cells in HeLa tumor spheroids before ( Figure 2A) and immediately after ( Figure 2B) treatment with membrane-impermeable Trypan Blue, which is capable of quenching of emission of fluorophores in the range of 500-600 nm (36). The fluorescence of the SNARF-pHLIP in this region was completely quenched indicating that SNARF is located in the extracellular space. The spectra of the SNARF-pHLIP before and after addition of Trypan Blue are shown on Figure 2C (the emission at 680 nm is associated with Trypan Blue). The bright field images of cells indicated that the vast majority of cells were viable.
A critical step is the identification of calibration curves to transfer 595/645 ratio values into pH values. The ratio of emission at 595 nm to 645 nm was calculated from the fluorescence spectra of the SNARF-pHLIP treated with POPC liposomes at various pHs, which were recorded under microscope. The ratios were used to establish a calibration curve, since pH in bulk of the solution, at the surface of liposomes, where most of the SNARF-pHLIP is located, and even inside a liposome are equilibrated quickly (25,37). We could not exclude the possibility that the SNARF signal and thus, calibration curve, might be different when the SNARF-pHLIP is located at the surface of real cells, which would not be unexpected given that nigericin calibrated intracellular SNARF curves differ from that of SNARF in solution(38)Therefore, we Thus, equations (1) and (2) will be used for the processing of fluorescence ratios obtained from spectra and images, respectively. The developed tool was applied to establish pH at the surface of metastatic (HeLa and M4A4) and non-metastatic (NM2C5) cancer cells grown in tumor spheroids in presence of 50 mM glucose, which enhances and promotes cellular metabolism ( Figure 3C). It is important to outline that the pH at the surface of metastatic cancer cells does not increase more than value of 7.0 even when the pH of bulk solution is around 7.9. Non-metastatic cancer cells are less acidic compared to metastatic, especially in the range of normal pH values. With a decrease of pH we observed equilibration of cell-surface pHs and bulk pH of media.
When the pH of media is less than 6.4, the pH at the surface of cancer cells in average did not decrease accordingly and did not dip below 6.35. The images were analyzed by the CFA program, which establishes the pH of the most bright cells. This data was correlated well with the results obtained by spectra analysis.
The advantage of our approach is in its applicability for pH measurements in vivo, since pHLIP can target acidic diseased tissue and tether imaging agents, including fluorescent, to the surface of cells (27). To validate this approach in vivo, we grew metastatic, HeLa and M4A4, and less metastatic, NM2C5 tumors in mice. When the tumors reached about 5-8 mm in diameter, the mouse was placed in condition of starvation for 24 hours in order to reduce flux of glucose to cancer cells from blood and increase pH in the tumor as much as possible, followed by single IV injection of the SNARF-pHLIP construct. At 4 hours post-injection, the mouse was anesthetized and the skin was removed from the tumor site. Fluorescent spectra and images were recorded from the tumor surface (the image is shown in Figure 4A). Then, the mouse obtained a single IP injection of solution of glucose. It was shown previously that the average extracellular pH decreases after glucose administration and reaches a minimum level 0.3 pH units below the initial value (40). We observed spectra changes after 40 minutes post-injection of glucose ( Figure 4B), no further spectral changes occurred after 40 minutes, which indicated acidification of tumor as monitored by our approach. Finally, the animal was euthanized, tumor was excised, cut in half and fluorescence was recorded from the center of the tumor. Figure 4C represents the mean of the surface pHs in tumor surface before and after glucose injection and in the center of the tumor. HeLa tumors are the most acidic even after 24h starvation period.
The mean values of surface pH in the center of HeLa, M4A4 and NM2C5 tumors are 6.51±0.22, 6.68±0.41 and 6.94±0.29, respectively with some HeLa tumors having pH as low as pH 6.1. M4A4 and NM2C5 tumors had similar pH before glucose injection, while pH was reduced more significantly in metastatic M4A4 tumor compared to non-metastatic NM2C5 tumors after glucose injection. Finally, we performed analysis of tumor tissues excised from mice and immediately treated with the SNARF-pHLIP ex vivo for 1 hour followed extensive washing and imaging SNARF fluorescence at 580 nm and 640 nm. Treatment was performed in PBS of pH7.4 in absence and presence of glucose. Glucose in solution promotes cellular metabolism selectively in glycolytic, highly metastatic cancer cells and enhances acidity near their surfaces. Thus, pHLIP preferentially inserts into plasma membrane of cells with low pH at the surface, such as cancer cells. At the same time, glucose does not affect significantly non-glycolytic cells in healthy tissue, which has normal surface pH (41). In Figure 4D we demonstrate the mean values of the surface pHs in highly metastatic human HeLa and murine 4T1 mammary tumor samples before and after treatment with glucose. The surface pHs dropped on 0.2 and 0.6 pH units from pH 6.7±0.3 to pH 6.5±0.4 and from pH 6.8±0.2 to pH 6.2±0.2 in HeLa and 4T1 tumor samples, respectively.

Discussion
Hypoxic conditions induce in a cell switch from the oxidative-phosphorylative mechanism of energy production to the glycolytic mechanism. In addition, malignant cancers have an elevated glucose uptake even under normal oxygen conditions, known as "aerobic glycolysis" or the Warburg effect (42)(43)(44). Glycolysis results in much higher level of the production of H + and lactic acid, the byproducts, which are readily pumped across a plasma membrane into the extracellular space and accumulate there, in poor-perfused regions such as solid tumor and ischemic stroke (45)(46)(47). Another contributor to extracellular acidity is associated with the expression of the carbonic anhydrase enzymes on the tumor cell surface, which catalyze the extracellular trapping of acid by hydrating cell-generated CO 2 into HCO 3 − and H + (48,49). All these mechanisms contribute towards an acidic extracellular milieu favoring diseases development and progressions. The extracellular pH of solid tumors plays one of the essential roles in almost all steps of metastasis: more acidic tumors became highly aggressive and metastatic (50). It was shown that the pH near the cell surface is the lowest and acidity decays with distance from a cell (13).