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. 2024 Feb;13(3):e6812.
doi: 10.1002/cam4.6812. Epub 2024 Jan 18.

Tumor therapy by targeting extracellular hydroxyapatite using novel drugs: A paradigm shift

Mohammed N Tantawy  1   2 , J Oliver McIntyre  1   2   3 , Fiona Yull  3   4 , M Wade Calcutt  5   6 , Dmitry S Koktysh  7   8 , Andrew J Wilson  4 , Zhongliang Zu  1   2 , Jeff Nyman  9   10 , Julie Rhoades  10   11 , Todd E Peterson  1   2 , Daniel Colvin  1 , Lisa J McCawley  9 , Jerri M Rook  3 , Barbara Fingleton  3 , Marta Ann Crispens  4   12 , Ronald D Alvarez  4 , John C Gore  1   2
Affiliations

Tumor therapy by targeting extracellular hydroxyapatite using novel drugs: A paradigm shift

Mohammed N Tantawy et al. Cancer Med. 2024 Feb.

Abstract

Background: It has been shown that tumor microenvironment (TME) hydroxyapatite (HAP) is typically associated with many malignancies and plays a role in tumor progression and growth. Additionally, acidosis in the TME has been reported to play a key role in selecting for a more aggressive tumor phenotype, drug resistance and desensitization to immunotherapy for many types of cancers. TME-HAP is an attractive target for tumor detection and treatment development since HAP is generally absent from normal soft tissue. We provide strong evidence that dissolution of hydroxyapatite (HAP) within the tumor microenvironment (TME-HAP) using a novel therapeutic can be used to kill cancer cells both in vitro and in vivo with minimal adverse effects.

Methods: We developed an injectable cation exchange nano particulate sulfonated polystyrene solution (NSPS) that we engineered to dissolve TME-HAP, inducing localized acute alkalosis and inhibition of tumor growth and glucose metabolism. This was evaluated in cell culture using 4T1, MDA-MB-231 triple negative breast cancer cells, MCF10 normal breast cells, and H292 lung cancer cells, and in vivo using orthotopic mouse models of cancer that contained detectable microenvironment HAP including breast (MMTV-Neu, 4T1, and MDA-MB-231), prostate (PC3) and colon (HCA7) cancer using 18 F-NaF for HAP and 18 F-FDG for glucose metabolism with PET imaging. On the other hand, H292 lung tumor cells that lacked detectable microenvironment HAP and MCF10a normal breast cells that do not produce HAP served as negative controls. Tumor microenvironment pH levels following injection of NSPS were evaluated via Chemical Exchange Saturation (CEST) MRI and via ex vivo methods.

Results: Within 24 h of adding the small concentration of 1X of NSPS (~7 μM), we observed significant tumor cell death (~ 10%, p < 0.05) in 4T1 and MDA-MB-231 cell cultures that contain HAP but ⟨2% in H292 and MCF10a cells that lack detectable HAP and in controls. Using CEST MRI, we found extracellular pH (pHe) in the 4T1 breast tumors, located in the mammary fat pad, to increase by nearly 10% from baseline before gradually receding back to baseline during the first hour post NSPS administration. in the tumors that contained TME-HAP in mouse models, MMTV-Neu, 4T1, and MDA-MB-231, PC3, and HCA7, there was a significant reduction (p<0.05) in 18 F-Na Fuptake post NSPS treatment as expected; 18 F- uptake in the tumor = 3.8 ± 0.5 %ID/g (percent of the injected dose per gram) at baseline compared to 1.8 ±0.5 %ID/g following one-time treatment with 100 mg/kg NSPS. Of similar importance, is that 18 F-FDG uptake in the tumors was reduced by more than 75% compared to baseline within 24 h of treatment with one-time NSPS which persisted for at least one week. Additionally, tumor growth was significantly slower (p < 0.05) in the mice treated with one-time NSPS. Toxicity showed no evidence of any adverse effects, a finding attributed to the absence of HAP in normal soft tissue and to our therapeutic NSPS having limited penetration to access HAP within skeletal bone.

Conclusion: Dissolution of TME-HAP using our novel NSPS has the potential to provide a new treatment paradigm to enhance the management of cancer patients with poor prognosis.

Keywords: PET; SPECT; breast cancer; hydroxyapatite; tumor extracellular pH; tumor metabolism; tumor microenvironment.

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Figures

FIGURE 1
FIGURE 1
Predicted structures of major components of NSPS. Analytical data (mass spectroscopy) along with FT‐IR and NMR (shown in supplementary data) suggest vinyl benzenesulfonate (C8H7O3S− [M‐H] exact mass 183.0125) and hydroxylated vinyl benzenesulfonate (C8H7O4S− [M‐H] exact mass 199.0074) as predominant components of NSPS.
FIGURE 2
FIGURE 2
Structure of the novel NSPS monomer. Structure and corresponding 1H NMR spectrum of the small molecule VU0945652 formulated at Vanderbilt University and postulated to be the active ingredient of NSPS.
FIGURE 3
FIGURE 3
NSPS leads to tumor cell death in cells with HAP. Dead to total cell ratio of 4 T1, MDA‐MB‐231 tumorigenic breast cell lines (both deposit HAP in their extracellular matrix), H292 lung tumor cell line which had no detectable ECM‐HAP, and MCF10 normal breast cell line that lacks extracellular HAP. All cells treated with 7 μM NSPS and counted within 18–24 h. * p < 0.0001; t = 33.2, df = 16; ** p < 0.0001; t = 23.7, df = 16; *** p = 0.0003, t = 4.6, df = 16; MCF10a; p = 0.3464; t = 0.97, df = 16.
FIGURE 4
FIGURE 4
VU0945652 is the active ingredient of NSPS polymer and tumor cell death is dose dependent. (Left) Impact of 20 mg of VU0945652 on 4 T1 breast and H292 lung tumor cultures. (Right) Dose effects of NSPS polymer (1X = 2.4 mg) and the small monomer VU0945652 (1X = 20 mg) on 4 T1 breast cells cultured in osteogenic cocktail medium. Controls are cell cultures that received saline with culture media. † p < 0.0001; t = 22.22, df = 6; †† p < 0.0001; t = 9.99, df = 6; * p < 0.0001; t = 31.01, df = 6; ** p = 0.0025; t = 5.000, df = 6; *** p < 0.0001; t = 18.42, df = 6; **** p < 0.0001; t = 12.25, df = 6.
FIGURE 5
FIGURE 5
NSPS induces acute alkalosis localized to the tumor. Panels A‐C are typical data from a single NSPS‐treated mouse bearing a 4 T1 tumor in the mammary fat pad. (A) Sample T2 weighed MRI spectrum of a 4 T1 breast tumor in the mammary fat pad of a white female Balb/c mouse. Regions of interest were drawn around the tumor. (B) CEST data acquired after injection of iohexol in the tumor (baseline or scan 0), just after i.v. injection of 25 mg/kg NSPS and every 7.25 min for a total of 10 scans post NSPS treatment. (C) Lorentzian fit of the tumor CEST data around the 4.3 ppm peak. (D) Changes in tumor extracellular pH compared to baseline (scan 0) is the average of 4 independent measurements per group (NSPS vs vehicle saline injected controls) on 4 T1 tumor bearing mice. Data points displayed as means ± SE.
FIGURE 6
FIGURE 6
NSPS increases tumor pH in vivo. Groups of mice bearing 4 T1 tumors in the mammary fat pad received 25 mg/kg NSPS (i.v.), vehicle (saline), or nothing at all (shams); n = 3 per cohort. The tumors were then homogenized and the overall (macro) pH of tumors that received NSPS was significantly higher (p < 0.05) than that of the other two groups. * p = 0.0031, t = 6.40, df = 4; ** p = 0.0064, t = 5.23, df = 4; sham vs saline: p = 0.8359, t = 0.22, df = 4.
FIGURE 7
FIGURE 7
NSPS dissolves TME‐HAP and inhibits glucose metabolism for up to 1 week. Representative images of a MMTV‐Neu breast tumors in the mammary fat pad of female FVP/n mice imaged with 18F‐NaF at (A) baseline (white arrow points to tumor) and (B) within 48 h following i.v. injection of one‐time 100 mg/kg of NSPS (see Table 1 for quantification and statistics). Approximately 24 h following baseline 18F‐NaF imaging, the mice were imaged with 18F‐FDG PET at (C) baseline, (D) 24 h post NSPS treatment and (E) 1 week post NSPS treatment. (F) Same mouse model imaged with FDG PET at baseline and (G) 1 week post vehicle (saline) injections. All tumors from all mice were harvested after the 1‐week FDG scan and underwent IHC analyses. The tumors of the NSPS‐treated mice tested positive for cleaved caspase 3 (apoptosis) (H) throughout the tumors and negative for Ki 67 (proliferation) (J). Some HAP was observed in the tumor via von Kossa (K) and alizarin red S (L) staining. Black arrows point to positive (black) stains. This is consistent with 18F‐NaF uptake in the tumor following treatment with NSPS. The vehicle treated mice were completely negative for cleaved caspase 3 (M) and positive for Ki 67 (N).
FIGURE 8
FIGURE 8
NSPS has minimal impact on tumors lacking detectable extracellular HAP. Representative image of a xenograft model of H292 lung tumor imaged with (A) 18F‐NaF PET but the tumor was not detected with this radiotracer. White arrows point to where the tumor should be. (B) Same mouse imaged with 18F‐FDG PET at baseline and (C) 24 h post treatment with 100 mg/kg NSPS. No changes in FDG uptake in the tumor were detected; 18F‐FDG uptake in the tumor at baseline and post NSPS was 2.3 ± 0.5 %ID/g in both cases. (D) Sections of harvested tumors which did not reveal positive cleaved caspase 3 but were positive for Ki 67 throughout the tumor (E). The tumors also tested negative for (F) von Kossa and (G) alizarin red S staining indicating absence of detectable TME‐HAP. Thus, this H292 mouse model serves as an in vivo negative control for testing NSPS.
FIGURE 9
FIGURE 9
One‐time NSPS significantly inhibits tumor growth rate (tumor indolence). Growth curves of MDA‐MB‐231 xenograft breast tumors in female athymic nu/nu mice. Data were fitted with exponential growth curves (dashed lines). See text for fit parameters.
FIGURE 10
FIGURE 10
NSPS has limited impact on skeletal bone. Three‐dimensional microCT images of femur bones harvested from age‐matched white balb/c mice. Red streaks on bone are the pores. The mice received either 100 mg/kg one‐time NSPS (left) or vehicle saline (right) 10 days before harvesting. The bone specimen were imaged in a microCT at a nominal resolution of 6 μm.
FIGURE 11
FIGURE 11
No evidence of nephrotoxicity following treatment with NSPS. Mice imaged with (A) 99mTc‐MAG3 SPECT (for renal function) and (B) with CT following an i.p. injection of Optiray CT contrast The images were taken 24–48 h after treatment with 100 mg/kg of NSPS. Normal renal function detected in both images.
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References

    1. Haka AS, Shafer‐Peltier KE, Fitzmaurice M, Crowe J, Dasari RR, Feld MS. Identifying microcalcifications in benign and malignant breast lesions by probing differences in their chemical composition using Raman spectroscopy. Cancer Res. 2002;62(18):5375‐5380. - PubMed
    1. Baker R, Rogers KD, Shepard N, Stone N. New relationships between breast microcalcifications and cancer. Br J Cancer. 2010;103(7):1034‐1039. - PMC - PubMed
    1. Cox RF, Hernandez‐Santana A, Ramdass S, McMahon G, Harmey JH, Morgan MP. Microcalcifications in breast cancer: novel insights into the molecular mechanism and functional consequence of mammary mineralisation. Br J Cancer. 2012;106(3):525‐537. - PMC - PubMed
    1. Morgan MP, Cooke MM, McCarthy GM. Microcalcifications associated with breast cancer: an epiphenomenon or biologically significant feature of selected tumors? J Mammary Gland Biol Neoplasia. 2005;10(2):181‐187. - PubMed
    1. Frappart L, Boudeulle M, Boumendil J, et al. Structure and composition of microcalcifications in benign and malignant lesions of the breast: study by light microscopy, transmission and scanning electron microscopy, microprobe analysis, and X‐ray diffraction. Human Pathology. 1984;15(9):880‐889. - PubMed

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