One cisplatin dose provides durable stimulation of anti-tumor immunity and alleviates anti-PD-1 resistance in an intraductal model for triple-negative breast cancer

Abstract

Aggressive triple-negative breast cancer (TNBC) is classically treated with chemotherapy. Besides direct tumor cell killing, some chemotherapeutics such as cisplatin provide additional disease reduction through stimulation of anti-tumor immunity. The cisplatin-induced immunomodulation in TNBC was here investigated in-depth using immunocompetent intraductal mouse models. Upon primary tumor transition to invasive carcinoma, cisplatin was injected systemically and significantly reduced tumor progression. Flow cytometric immunophenotyping was corroborated by immunohistochemical analyses and revealed both differential immune cell compositions and positivity for their programmed death (PD)-1 and PD-ligand (L)1 markers across body compartments, including the primary tumor, axillary lymph nodes and spleen. As key findings, a significant decrease in immunosuppressive and a concomitant increase in anti-tumor lymphocytic cell numbers were observed in the axillary lymph nodes and spleen, highlighting their importance in cisplatin-stimulated anti-tumor immunity. These immunomodulatory effects were already established following the first cisplatin dose, indicating that early cisplatin-mediated events may determine (immuno)therapeutic outcome. Furthermore, a single cisplatin dose sufficed to alleviate anti-PD-1 resistance in a 4T1-based model, providing add-on disease reduction without toxic side effects as seen upon multiple cisplatin dosing. Overall, these results highlight cisplatin as immunotherapeutic ally in TNBC, providing durable immunostimulation, even after a single dose.

Chitin-mediated blockade of chitinase-like proteins reduces tumor immunosuppression, inhibits lymphatic metastasis and enhances anti-PD-1 efficacy in complementary TNBC models

“…Firefly luciferase-expressing 4T1 (4T1-luc) mammary tumor cells which resemble metastatic human TNBC were provided as a kind gift from Prof. Clare Isacke (Breakthrough Breast Cancer Research Centre, London, UK). The 4T1-luc cells were cultured as previously described [ 22 ]. Firefly luciferase-expressing 66cl4 (66cl4-luc) cells were kindly provided by Prof. Traci R. Lyons (University of Colorado Anschutz Medical Campus, Denver, CO, USA).…”

“…Female BALB/c mice of 8 weeks (w) old were mated with male BALB/c mice of similar age and gave birth to pups within 3 w. The pups were weaned 12–14 days (d) later, after which the individually caged, lactating mother mice were intraductally inoculated in the third mammary gland pair with 5 × 10 4 4T1-luc or 1–2 × 10 5 66cl4-luc cells suspended in a cold mixture of PBS and Matrigel® (1:10; Corning, Bedford, MA, USA). The intraductal inoculations were conducted using a 32G pediatric needle under isoflurane inhalation anesthesia and buprenorphine analgesia as described previously [ 22 ].…”

“…Anti-mPD-1-mIgG1e3 InvivoFit™ (Invivogen) derived from clone RMP1-14 and anti-β-Gal-mIgG1e3 InvivoFit™ (Invivogen) clone T9C6 were used for anti-PD-1 ICB and isotype control, respectively, through i.p. injection at 25 µg or 200 µg/mouse as previously described [ 22 ]. Anti-CHI3L1 clone 4E3-F2 (M3; Absolute Antibody, Oxford, UK) and anti-Fluorescein clone 4–4-20 (Absolute Antibody) were used for CHI3L1 blockade and isotype control, respectively, through i.p.…”

“…For the in vivo imaging, tumor-bearing mice were i.p. injected with 200 µl D-luciferin (20 mg/ml PBS; PerkinElmer, Zaventem, Belgium) and caudally placed in the IVIS Lumina III system (PerkinElmer) under isoflurane inhalation anesthesia as described previously [ 22 ]. For the ex vivo imaging of metastases, tumor-bearing mice were terminally sedated and sacrificed through cervical dislocation, after which axillary lymph nodes, lungs and spleen were quickly isolated and bioluminescent signals were captured with the IVIS system.…”

“…Primary tumors, axillary lymph nodes and spleens were isolated from untreated and treated mice upon sacrification. Primary tumors were digested into single cell suspensions using a commercially available tumor dissociation kit and gentleMACS Dissociator with accompanying C tubes (Miltenyi Biotec, Leiden, The Netherlands) as previously described [ 22 ]. Manual processing of axillary lymph nodes and spleens into single cell suspensions was also performed as previously described [ 22 ].…”