Advancements in monitoring and predicting of patient-specific response of triple negative breast cancer (TNBC) to immunotherapy (IMT) with and without chemotherapy are needed. Using granzyme B-specific positron emission tomography (GZP-PET) imaging, we aimed to monitor changes in effector cell activation in response to IMT with chemotherapy in TNBC. TNBC mouse models received the paclitaxel (PTX) ± immune checkpoint inhibitors anti-programmed death 1 (anti-PD1) and anti-cytotoxic T-lymphocyte 4 (anti-CTLA4). GZP-PET imaging was performed on treatment days 0, 3, and 6. Mean standard uptake value (SUV_mean), effector cell fractions, and SUV histograms were compared. Mice were sacrificed at early imaging timepoints for cytokine and histological analyses. GZP-PET imaging data revealed differences prior to tumor volume changes. By day six, responders had SUV_mean ≥ 2.2-fold higher (p < 0.0037) and effector cell fractions ≥ 1.9-fold higher (p = 0.03) compared to non-responders. IMT/PTX resulted in a significantly different SUV distribution compared to control, indicating broader distribution of activated intratumoral T-cells. IMT/PTX resulted in significantly more necrotic tumor tissue and increased levels of IL-2, 4, and 12 compared to control. Results implicate immunogenic cell death through upregulation of key Th1/Th2 cytokines by IMT/PTX. Noninvasive PET imaging can provide data on the TNBC tumor microenvironment, specifically intratumoral effector cell activation, predicting response to IMT plus chemotherapy.

Hypoxia is a common feature of the tumor microenvironment, including that of triple-negative breast cancer (TNBC), an aggressive breast cancer subtype with a high five-year mortality rate. Using [¹⁸F]-fluoromisonidazole (FMISO) positron emission tomography (PET) imaging, we aimed to monitor changes in response to immunotherapy (IMT) with chemotherapy in TNBC. TNBC-tumor-bearing mice received paclitaxel (PTX) ± immune checkpoint inhibitors anti-programmed death 1 and anti-cytotoxic T-lymphocyte 4. FMISO-PET imaging was performed on treatment days 0, 6, and 12. Max and mean standard uptake values (SUV_max and SUV_mean, respectively), histological analyses, and flow cytometry results were compared. FMISO-PET imaging revealed differences in tumor biology between treatment groups prior to tumor volume changes. 4T1 responders showed SUV_mean 1.6-fold lower (p = 0.02) and 1.8-fold lower (p = 0.02) than non-responders on days 6 and 12, respectively. E0771 responders showed SUV_mean 3.6-fold lower (p = 0.001) and 2.7-fold lower (p = 0.03) than non-responders on days 6 and 12, respectively. Immunohistochemical analyses revealed IMT plus PTX decreased hypoxia and proliferation and increased vascularity compared to control. Combination IMT/PTX recovered the loss of CD4+ T-cells observed with single-agent therapies. PET imaging can provide timely, longitudinal data on the TNBC tumor microenvironment, specifically intratumoral hypoxia, predicting therapeutic response to IMT plus chemotherapy.