MC38 同系移植瘤模型（C57BL/6，文献参考）

达到可测量体积（Day 7-10 左右）后按肿瘤体积分层随机分组，每组 8-10 只（来源 Part B）。

获得可供光学显微镜观察的 H&E 染色组织切片：细胞核蓝紫色、细胞质与结缔组织纤维粉红色，用于观察组织中细胞的整体结构与形态细节。本方案本身不产出病理评分数值，也不提供特定分子标志物的定量结果。

获得同系移植瘤生长与应答数据：肿瘤生长曲线（V=(长×宽²)/2）、肿瘤生长抑制 TGI=(1-治疗组均体积/同型对照均体积)×100%（大于 60% 视为生物学显著）、体重变化与生存观察；终点取材进行肿瘤称重、10% 福尔马林固定 IHC、解离流式（肿瘤浸润淋巴细胞）与血清细胞因子分析（来源 Part D/E 读出）。

脱蜡（Dewaxing） 石蜡切片置于二甲苯中脱蜡 3 次，每次 00:03:00。 复水（Rehydration） 石蜡切片依次置于 100% 乙醇中 3 次、每次 00:03:00，再置于 95% 乙醇中 1 次、00:03:00，随后用水漂洗。 苏木素核染色（Hematoxylin staining） 石蜡切片以苏木素染液染色 00:00:20，随后在流水下冲洗 00:01:00。 分化与返蓝（Differentiation and bluing） 石蜡切片置于返蓝液中 00:01:00，随后在流水下冲洗 00:01:00；此步使细胞核呈现蓝色。 伊红染色（Eosin staining） 石蜡切片以伊红复染 00:00:10；此步使细胞质成分与结缔组织纤维呈现粉红色。 脱水与透明（Dehydration and Clearing） 石蜡切片依次置于 95% 乙醇中 00:02:00、100% 乙醇中 2 次、每次 00:02:00，随后置于二甲苯中 2 次、每次 00:02:00。 封片（Mounting） 石蜡切片以 Permount 封片介质加盖玻片封固。

**Part A — Tumor Cell Preparation** **Part A — Tumor Cell Preparation** **Step 1: Culture tumor cells.** **Step 1: Culture tumor cells.** Expand tumor cells in complete medium. Harvest at log-phase growth (70-80% confluence). Passage cells at least once after thawing before use in vivo — never implant directly from a thawed vial. **Step 2: Prepare cell suspension for implantation.** **Step 2: Prepare cell suspension for implantation.** Trypsinize cells, wash 2× with sterile PBS to remove serum. Count viable cells by trypan blue exclusion — viability must be >95%. Resuspend at the required density in sterile PBS (or 1:1 PBS:Matrigel for poorly engrafting lines). Keep cell suspension on ice. **NOTE:** Recommended cell numbers per mouse: MC38: 5 × 10⁵; CT26: 5 × 10⁵; B16-F10: 2 × 10⁵; 4T1: 1 × 10⁵. Adjust based on local experience with growth kinetics. **Step 3: Implant tumors (Day 0).** **Step 3: Implant tumors (Day 0).** Shave the right flank of each mouse. Disinfect with 70% ethanol. Inject 100 μL cell suspension subcutaneously using a 27G insulin syringe. Insert needle bevel-up, advance ~5 mm under the skin, inject slowly. A visible bleb confirms correct s.c. delivery. Avoid injecting into the muscle. **Part B — Tumor Monitoring and Randomization** **Part B — Tumor Monitoring and Randomization** **Step 4: Monitor tumor establishment (Day 0-7).** **Step 4: Monitor tumor establishment (Day 0-7).** Palpate injection sites daily from Day 5. Tumors typically become palpable by Day 5-7 (MC38, CT26) or Day 3-5 (B16-F10). Record the day each tumor becomes measurable. **Step 5: Measure tumors and randomize (Day 7-10).** **Step 5: Measure tumors and randomize (Day 7-10).** When average tumor volume reaches 50-100 mm³, measure tumors with digital calipers. Calculate volume using the formula: V = (length × width²) / 2, where length is the longest diameter and width is perpendicular to length. Randomize mice into treatment groups so that mean tumor volume is equal across groups. Exclude mice with tumors 150 mm³. **NOTE:** Randomization day is treatment Day 0. All subsequent dosing is referenced from this day, not from implantation day. **Part C — Antibody Dosing** **Part C — Antibody Dosing** **Step 6: Prepare antibody doses.** **Step 6: Prepare antibody doses.** Calculate the required dose per mouse based on body weight (typical: 200 μg/mouse or 10 mg/kg for anti-PD-1; 200 μg/mouse for anti-CTLA-4). Dilute InVivo-grade antibody in sterile endotoxin-free PBS to a final injection volume of 100-200 μL per mouse. Prepare isotype control at the same concentration and volume. **NOTE:** InVivo-grade antibodies must be low-endotoxin (<1 EU/mg), azide-free, and formulated in sterile PBS. Do not use research-grade antibodies containing sodium azide for in vivo injection — azide is toxic to animals. **Step 7: Administer antibodies (Treatment Day 0).** **Step 7: Administer antibodies (Treatment Day 0).** Inject each mouse intraperitoneally (i.p.) with 100-200 μL of antibody solution. Use a 27-29G insulin syringe. Restrain the mouse in a supine position, tilt head-down slightly, and inject into the lower right quadrant of the abdomen to avoid puncturing the cecum. **Step 8: Continue dosing schedule.** **Step 8: Continue dosing schedule.** Typical dosing schedules: Anti-PD-1 monotherapy: 200 μg i.p. on Days 0, 3, 6, 9 (total 4 doses, twice weekly). Anti-CTLA-4 monotherapy: 200 μg i.p. on Days 0, 3, 6 (total 3 doses). Combination (anti-PD-1 + anti-CTLA-4): anti-PD-1 200 μg + anti-CTLA-4 100 μg i.p. on Days 0, 3, 6, 9. Isotype control group: matched isotype antibody at the same dose and schedule. **NOTE:** Administer combination antibodies as separate injections (two i.p. injections) or pre-mix in the same syringe if compatibility has been verified. Do not exceed 400 μL total i.p. volume per injection in a mouse. **Part D — Efficacy Assessment** **Part D — Efficacy Assessment** **Step 9: Measure tumors.** **Step 9: Measure tumors.** Measure tumor dimensions with calipers every 2-3 days. Calculate volume: V = (length × width²) / 2. Record body weight simultaneously. **Step 10: Record data.** **Step 10: Record data.** Record individual tumor volumes, group means ± SEM, and body weight for each mouse at every measurement time point. **Step 11: Calculate tumor growth inhibition (TGI).** **Step 11: Calculate tumor growth inhibition (TGI).** TGI (%) = [1 - (mean tumor volume of treatment group / mean tumor volume of isotype control group)] × 100. TGI >60% is generally considered biologically significant. **Part E — Endpoint Criteria** **Part E — Endpoint Criteria** **Step 12: Apply humane endpoints.** **Step 12: Apply humane endpoints.** Euthanize individual mice when any of the following occur: tumor volume exceeds 2,000 mm³ (or institutional limit); tumor becomes ulcerated or necrotic; body weight loss exceeds 20% from baseline; animal shows signs of distress (hunched posture, labored breathing, immobility). Record time to endpoint for survival analysis. **Step 13: Terminal analysis.** **Step 13: Terminal analysis.** At study endpoint, harvest tumors for downstream analysis: weigh tumor, fix in 10% formalin for IHC, or dissociate for flow cytometry (immune cell profiling of tumor-infiltrating lymphocytes). Collect serum for cytokine analysis if needed.