Impact of Chemical-Induced Mutational Load Increase on Immune Checkpoint Therapy in Poorly Responsive Murine Tumors

Abstract

A recurring historic finding in cancer drug development is encouraging antitumor effects observed in tumor-bearing mice that fail to translate into the clinic. An intriguing exception to this pattern is immune checkpoint therapy, as the sustained tumor regressions observed in subsets of cancer patients are rare in mice. Reasoning that this may be due in part to relatively low mutational loads of mouse tumors, we mutagenized transplantable mouse tumor cell lines EMT-6/P, B16F1, RENCA, CT26, and MC38 with methylnitro-nitrosoguanidine (MNNG) or ethylmethane sulfonate (EMS) and tested their responsiveness to PD-L1 blockade. Exome sequencing confirmed an increase in somatic mutations by mutagen treatment, an effect mimicked in EMT-6 variants chronically exposed to cisplatin or cyclophosphamide. Certain mutagenized variants of B16F1, EMT-6/P, CT26, and MC38 (but not RENCA) were more immunogenic than their parents, yet anti-PD-L1 sensitization developed only in some EMT-6/P and B16F1 variants. Treatment response patterns corresponded with changes in immune cell infiltration and especially increases in CD8 T cells. Chronically cisplatin-exposed EMT-6 variants were also more responsive to anti-PD-L1 therapy. Although tumor PD-L1 expression was upregulated in chemotherapy-exposed variants, PD-L1 expression levels were not consistently associated with anti-PD-L1 treatment activity across mutagenized or chemotherapy-exposed variants. In summary, mutagenized and more immunogenic mouse tumors were not universally sensitized to PD-L1 blockade. Chemically mutagenized variants may be useful to evaluate the impact of immunologically "hot" or "cold" tumors with a high mutational load, to which certain chemotherapy agents may contribute, on immunotherapy outcomes..

Immunostimulatory and anti-tumor metronomic cyclophosphamide regimens assessed in primary orthotopic and metastatic murine breast cancer

“…6a). PD-L1 blockade monotherapy had modest anti-tumor effects similar to results obtained previously and significantly extended survival 35 (Fig. 6b).…”

“…We have previously shown that PD-L1 blockade can have therapeutic effects in the EMT6-CDDP tumor model when treating both primary tumors and postsurgical metastatic disease in the neoadjuvant or adjuvant treatment settings 35,36 . Due to the findings of increased PD-L1 expression after CTX treatment, as well as CyTOF data suggesting that there was an increased infiltration of CD4 + and CD8 + T cells seen with CTX140 1q6d therapy, this model was used for testing whether any of the CTX regimens in combination with PD-L1 blockade could enhance overall therapeutic effects.…”

Beneficial modulation of the tumor microenvironment and generation of anti-tumor responses by TLR9 agonist lefitolimod alone and in combination with checkpoint inhibitors

“…In the clinical setting, tumor burden at the start of treatment have an influence on the benefit from the treatment, with early treatment and smaller tumors being advantageous. 45,46 In keeping with this, we observed the expected augmented antitumor effect in a tumor model when inoculating a lower dose of CT26 cells for tumor growth ( Figure S3(a-c)). Furthermore, an earlier treatmentstarting as early as 8 days before tumor inoculationled to clearly better tumor inhibition ( Figure S3).…”

Therapeutic impact of Nintedanib with paclitaxel and/or a PD-L1 antibody in preclinical models of orthotopic primary or metastatic triple negative breast cancer

“…We therefore decided to evaluate whether combining the nintedanib and paclitaxel doublet with a PD-L1 immune checkpoint antibody could improve overall anti-tumor activity in the syngeneic immunocompetent EMT-6 and EMT-6/CDDP mouse tumor models. We observed that EMT-6/CDDP cells express higher levels of PD-L1 in vitro than the parental EMT-6 cells [79]. In this study, we found that the PD-L1 antibody treatment induced a similar tumor growth delay, with respect to the control groups, when treating either EMT-6 or EMT-6/CDDP primary tumors (Fig.…”

“…We have found that EMT-6/CDDP cell line is more aggressive and metastatic, particularly to the lungs, compared to the parental cell line (unpublished observations). Also, we recently reported that this cell line expresses much higher levels of PD-L1 in vitro compared to the drug-sensitive parental cell line (EMT-6) [79].…”