Atezolizumab

All cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA). The identities of the cell lines were verified by STR analysis, and the cell lines were confirmed to be mycoplasma free. The cells were maintained in DMEM with 10% foetal bovine serum. Cell culture media and supplements were obtained from Life Technologies, Inc. Recombinant human PD-L1/B7-H1 Fc chimaera protein was obtained from R&D Systems (Minneapolis, MN). Cetuximab was purchased from Merck. Trastuzumab and atezolizumab were purchased from Roche Ltd. Residues 1–619 of the extracellular domain of EGFR (EGFR-ECD) and residues 1–646 of HER2-ECD were prepared using the pcDNA3.4 expression vector (Invitrogen) and FreeStyle 293 expression system (Invitrogen)^{54 (link),55 (link)}. Information about the primary antibodies used in this work is included in Supplementary Table 2.

A CCK-8 assay (Sigma, USA) was used to assess the effect of ICB with and without FLOT chemotherapy regimen on the proliferation rate of OE33 cells and SK-GT-4 cells. 5 × 10³ OAC cells were adhered in a 96 well plate at 37 °C, 5% CO2 overnight. The media was removed, and cells were cultured for 48 h in complete RPMI in the absence or presence of nivolumab (10 μg/ml, Opdivo, Bristol-Myers Squibb, USA), atezolizumab (10 μg/ml, Tecentriq, Roche, USA), A2aR antagonist (3 μM, Bio-techne, USA) with and without the FLOT chemotherapy regimen (IC₅₀ dose). 5 μl of CCK-8 solution was added to each well, followed by a 1.5 h incubation in the dark at 37 °C, 5% CO2. The optical density at 450 nm and 650 nm (reference wavelength) was measured using the Versa Max microplate reader (Molecular Devices, Sunnyvale, CA, USA) to determine a viable cell number. All of the data were analysed from three independent experiments.

Tumor organoids were treated with Sorafenib, Cisplatin, Oxaliplatin and 5‐FU (MedChemExpress), and Atezolizumab (Roche) at indicated concentrations. PDO mixed with a culture medium containing 10% Matrigel was pumped into microchips using microsyringes at a controlled flow velocity (10 µL min⁻¹). After organoids reached around 200 µm diameter in size, drugs were carefully delivered into microchips using microsyringes, followed by another 6‐day culture. Organoids cultured on microchips were visualized by microscopy. For drug screening on 96‐well plates, cell viability was assessed by CCK‐8 assay (Glpbio, GK10001) or Annexin V‐FITC (ES Science) staining with flow cytometry, according to the manufacturer's instruction. Dose‐response curves were analyzed by GraphPad Prism software (Dotmatics), and the mean area of total PDOs in each microarray unit was analyzed by Image J software.

Cobimetinib (provided by Hoffmann-La Roche) is an oral selective inhibitor of the MEK pathway. Vemurafenib (provided by Hoffmann-La Roche) is a low-molecular-weight inhibitor of BRAF serine–threonine kinase. Atezolizumab (provided by Hoffmann-La Roche) is an anti-PD-L1 antibody.
The study will include three possible treatments: (1) Vemurafenib 960 mg twice daily per os from day 1 to 42; (2) Vemurafenib 720 mg twice daily per os from day 1 to 42; (3) Cobimetinib 60 mg once daily per os from day 1 to 21 and from day 29 to 42; Cobimetinib should not be taken on days 22–28; and (4) Atezolizumab 840 mg intravenous for two cycles (days 22 and 43).
Patients will be randomized to three different arms: A) BRAF-mutated patients will receive (1) + (3) for 6 weeks; B) BRAF-mutated patients will receive (2) + (3) + (4) for 6 weeks; C) BRAF WT patients will receive (3) + (4) for 6 weeks.
All patients will also receive Atezolizumab 1200 mg intravenous every 3 weeks for 17 cycles after surgery and after a second screening period (up to 6 weeks).

All patients were treated with first-, second-, or third-generation EGFR-TKIs prior to immune checkpoint inhibitors. For immunotherapy, patients were treated with one of the following anti-PD-1 or PD-L1 agents until disease progression, or unacceptable toxicity: pembrolizumab (Merck & Co., USA), nivolumab (Bristol-Myers Squibb, USA), sintilimab (Innovent Biologics, China), toripalimab (Shangha Merck & Co.), camrelizumab (Jiangsu Hengrui Medicine, China), tislelizumab (BeiGene, China), durvalumab (AstraZeneca, USA), or atezolizumab (Roche, USA). This study was approved by the institutional review board of Shandong Cancer Hospital and Institute and was performed in accordance with the Declaration of Helsinki. Individual consent for this retrospective analysis was waived.

TH1579 was prepared according to published methods (WO2015187088). Cisplatin was purchased from Sigma-Aldrich. Atezolizumab (Tecentriq, 1200 mg) was from Roche.