Cyclophosphamide

Enzyme linked immunosorbent assay (ELISA) kits for interleukin-10 (IL-10), interferon-gamma (IFN-γ), and immunoglobulins (IgA, IgG, and IgM) were obtained from eBioscience (San Diego, CA). Cyclophosphamide was obtained from Baxter Oncology GmbH. Other reagents were purchased from commercial suppliers without any treatments, unless otherwise noted.

In all experiments, groups of female, CD-1 mice, 19 to 21 g, were obtained from Charles River Laboratories. At a minimum, animals were injected subcutaneously with 40 mg/kg of body weight of triamcinolone acetonide (Kenalog-10; Bristol-Myers Squibb) on the day preceding fungal inoculation (day −1). On day 0, animals were anesthetized in an isoflurane chamber and inoculated intranasally with fungal conidia resuspended in 30 µl of phosphate-buffered saline (PBS). Conidial densities of the inocula varied between experiments and are specified in the figure legends. Moreover, the animals in the DCF-3 experiment (Fig. 4B) as well as the Af293 experiment shown in Fig. S7B in the supplemental material received a second 40-mg/kg injection on day +3. For the chemotherapy model (see Fig. S7C), animals received both the 40-mg/kg Kenalog injection on day −1 and a 150-mg/kg intraperitoneal injection of cyclophosphamide (Baxter Health Care Corporation) on days −1 and +3.
In all cases, moribund animals were euthanized by CO₂ asphyxiation. Mortality curves between groups were compared by the log rank test.

All animal studies were conducted in the animal facility at Innovent Biologics with protocols approved by the Institutional Animal Care and Use Committees at Innovent Biologics. In the Panc02-claudin 18.2 pancreatic tumor model, CD45.2⁺ C57BL/6 recipient mice (6–8 weeks old, female, Vital River, Beijing, China) were inoculated subcutaneously with 4 × 10⁶ tumor cells in the right flank on Day 9 or 11 in the absence or presence of lymphodepletion pretreatment, and when the average tumor volume reached 80–100 mm³, they received one dose of intravenous (i.v.) infusion of 2.5 or 5 × 10⁶ CAR-T cells prepared from CD45.1⁺ congenic mice of the C57BL/6 genetic background with cyclophosphamide (CPA; Baxter Oncology GmbH) intraperitoneally (i.p.) administered at a dose of 150 mg/kg 1 day before CAR-T-cell infusion. In the B16F10-claudin 18.2 melanoma model, mice were inoculated subcutaneously with 4 × 10⁵ tumor cells in the flank of C57BL/6 mice on Day 11; when the average tumor volume reached 150 mm³, CPA was administered i.p. 1 day before i.v. infusion of 5 × 10⁶ CAR-T cells. In all experiments, tumor growth was measured by calipers twice a week, tumor volumes were calculated using the formula V = (L × W2)/2, and tumor growth inhibition (TGI) was defined as 1 − mean (TV_treat)/mean (TV_control). The mice were followed up for survival in one experiment.

All animals were administered ketoconazole (Torrent pharmaceuticals, Indrad—382721, Dist. Mehsana, India) orally at a dose of 10 mg/kg in 0.5% solution of carboxymethylcellulose (Carboxymethylcellulose sodium salt, Sigma-Aldrich, St. Louis, MO, USA) and cyclosporine (TEVA Czech Industries, Opava, Czech Republic) at a dose of 30 mg/kg intraperitoneally for 7 days, from day 10 to day 4 prior to LLC injection. On days 3 and 1 prior to injection of LLC cells, cyclophosphamide was administered subcutaneously at a dose of 60 mg/kg (Baxter Oncology GmbH, Halle/Westfalen, Germany). To monitor the immunosuppression, white blood cell count (WBC) was calculated 1 day before the administration of the LLC cell suspension [40 (link)].

Chemoresistant BT-474 and BT-474 NRP-1 variant cell lines were generated in a similar protocol to that described previously [13 (link)]. Briefly, the cells were treated with four cycles of a combination of 0.5 uM of Doxorubicin (Brand name Adriamycin, Pharmacia, Italy) + 300 nM Cyclophosphamide (Brand name Cytoxan, Baxter, Germany) (cells will be referred to as 4xAC) followed by four cycles of 20 nM Paclitaxel (Brand name Taxol, EBEWE Pharma, Austria) (cells will be referred to as 4xAC + 4xPAC). Each cycle was for a duration of 72 h followed by a recovery period until confluency was achieved prior to commencement of the next cycle. Protein lysate and RNA was extracted from the 4xAC and 4xAC + 4xPAC resistant cell lines and stored at − 80 °C.