Introduction

Not all cancer patients respond to immune checkpoint blockade therapy (ICB). Several factors can cause immunotherapy resistance, e.g., mutation load, upregulation of alternative checkpoints, and extracellular acidosis.¹ In this research, extracellular tumor acidosis is targeted to reverse the immunotherapy resistance.² The murine melanoma B16-F4 model³ which is resistant to immunotherapy is employed to test the hypothesis. The extracellular tumor acidosis can be harnessed by inhibiting the Vacuolar ATPase (VATPase) that pumps the proton out of the cell; monocarboxylate transporter 1 (MCT1) that transports lactic acid intracellular to extracellular surface; and Carbonic Anhydrase IX (CA-IX) that neutralizes intracellular acids and causes extracellular acidosis. The inhibitors used to block the activity of VATPase, MCT-1 and CA-IX are esomeprazole, AZD3965, and acetazolamide respectively. The altered pH due to treatment with pH sensitizers are tracked using acidoCEST MRI (extracellular pH (pHe)) and altered glycolysis due to ICB treatment are interrogated employing 1-¹³C Pyruvate HP-MRI.

Methods

The extracellular pH of immunotherapy resistant melanoma B16-F4 mouse tumor was modulated using IP injections of esomeprazole to make them sensitive to ICB by modulating the pHe and followed by the anti-CTLA4 and anti-PD1 injections. The mice treated with PBS and PBS + ICB are control cohorts. There was total 24 mice categorized into four groups of PBS, esomeprazole, PBS + ICB, and esomeprazole + ICB treated. The acidoCEST MRI was acquired day after the final treatment and HP-MRI data was acquired two days after the final treatment. All animal tissues were collected were snap frozen in liquid nitrogen and stored in -80 ⁰C refrigerator for further ex-vivo NMR-based metabolomics and RNA-seq study.

Results

Our initial results with inhibition of VATPase by Esomeprazole followed by ICB treatment after 24 hours didn’t reveal significant changes in pyruvate to lactate conversion compared to PBS injections followed by ICB treatment in B16-F4 mice. In agreement with HP- MRI data, pHe also didn’t exhibit any significant difference between the groups of esomeprazole + ICB and PBS + ICB treatments. Esomeprazole inhibits VATPAse that pumps proton out of the tumor cell, hence contributing to maintain the intracellular pH and acidifying the extracellular matrix. The idea was to increase the pHe by blocking the VATPase and hence providing favorable environment for the immune cells to act on tumor. However, the esomeprazole didn’t alter the pHe significantly in immunotherapy resistant B16-F4 melanoma model and we didn’t observe much beneficial change in survival curve with combination treatment of esomeprazole + ICB treatment compared to PBS + ICB.

Conclusion

The pH was not altered significantly in combined treatment of esomeprazole and ICB compared to PBS+ICB in immunotherapy resistant B16-F4 melanoma mouse model. Modification of the extracellular pH is being tested with MCT-1 inhibitor, AZD3965 and CA-IX inhibitor, acetazolamide.  

References:

1. Huber, V.;  Camisaschi, C.;  Berzi, A.;  Ferro, S.;  et al., In Cancer acidity: An ultimate frontier of tumor immune escape and a novel target of immunomodulation. Seminars in Cancer Biology, Elsevier: 2017; pp 74-89.  

 2. Scott, K. E.; Cleveland, J. L., Lactate wreaks havoc on tumor-infiltrating T and NK cells. Cell Metabolism 2016, 24 (5), 649-650.

3. Jaiswal, A. R.;  Liu, A. J.;  Pudakalakatti, S.;  Dutta, P.;  et al., Melanoma Evolves Complete Immunotherapy Resistance through the Acquisition of a Hypermetabolic PhenotypeCancer Hypermetabolism Confers Immunotherapy Resistance. Cancer Immunology Research 2020, 8 (11), 1365-1380.