The interplay of positive and negative interactions between drug-sensitive and resistant cells influences the effectiveness of treatment in heterogeneous cancer cell populations. Here, we study interactions between estrogen receptor-positive breast cancer cell lineages that are sensitive and resistant to ribociclib-induced cyclin-dependent kinase 4 and 6 (CDK4/6) inhibition. In mono- and coculture, we find that sensitive cells grow and compete more effectively in the absence of treatment. During treatment with ribociclib, sensitive cells survive and proliferate better when grown together with resistant cells than when grown in monoculture, termed facilitation in ecology. Molecular, protein, and genomic analyses show that resistant cells increase metabolism and production of estradiol, a highly active estrogen metabolite, and increase estrogen signaling in sensitive cells to promote facilitation in coculture. Adding estradiol in monoculture provides sensitive cells with increased resistance to therapy and cancels facilitation in coculture. Under partial inhibition of estrogen signaling through low-dose endocrine therapy, estradiol supplied by resistant cells facilitates sensitive cell growth. However, a more complete blockade of estrogen signaling, through higher-dose endocrine therapy, diminished the facilitative growth of sensitive cells. Mathematical modeling quantifies the strength of competition and facilitation during CDK4/6 inhibition and predicts that blocking facilitation has the potential to control both resistant and sensitive cancer cell populations and inhibit the emergence of a refractory population during cell cycle therapy.

PD-1 blockade unleashes the potent antitumor activity of CD8 cells but can also promote immunosuppressive T regulatory (Treg) cells, which may worsen response to immunotherapy. Tumor Treg inhibition is a promising strategy to overcome therapeutic resistance; however, the mechanisms supporting tumor Tregs during PD-1 immunotherapy are largely unexplored. Here, we report that PD-1 blockade increases tumor Tregs in mouse models of immunogenic tumors, including melanoma, and metastatic melanoma patients. Unexpectedly, Treg accumulation was not caused by Treg-intrinsic inhibition of PD-1 signaling but instead depended on an indirect effect of activated CD8 cells. CD8 cells colocalized with Tregs within tumors and produced IL-2, especially after PD-1 immunotherapy. IL-2 upregulated the anti-apoptotic protein ICOS on tumor Tregs, causing their accumulation. ICOS signaling inhibition before PD-1 immunotherapy resulted in increased control of immunogenic melanoma. Thus, interrupting the intratumor CD8:Treg crosstalk is a novel strategy that may enhance the efficacy of immunotherapy in patients.

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