Briefly, surgically resected tumors were maintained in DMEM-F12 (Gibco) supplemented with 1% HEPES (Sigma-Aldrich), 1% L-glutamine (Gibco), 10% FBS (Gibco), 2% penicillin/streptomycin (Sigma-Aldrich), and 10 uM Y-27632 (R&D Systems). Tumors were digested with collagenase solution (5 mL of the above medium with 75 uL collagenase, 124 ug/mL dispase type II, and 0.2% Primocen) for 30 min and then filtered through a 70 um filter (Corning). An organoid pellet was obtained by centrifugation (200x g for 10 min). Organoids were suspended in Matrigel (Corning, Tehama County, CA) with IntestiCult Organoid Growth Medium (#06010, STEMCELL Technologies) and seeded in 12-well plates. Approximately 750 uL of IntestiCult Organoid Growth Medium was added to each well. Organoids were divided into five groups of control, curcumin (3.0 ug/mL), andrographis (30.0 ug/mL), their combination (curcumin; 3.0 ug/mL, andrographis; 30.0 ug/mL), and their combination plus ferrostatin-1 (curcumin; 3.0 ug/mL, andrographis; 30.0 ug/mL; ferrostatin-1; 20 uM). Following forty-eight hours of treatment, the numbers of organoids (<100 um) and their mean sizes were examined using Image J software.

Briefly, surgically resected tumors were maintained in DMEM-F12 (Gibco) supplemented with 1% HEPES (Sigma-Aldrich), 1% L-glutamine (Gibco), 10% FBS (Gibco), 2% penicillin/streptomycin (Sigma-Aldrich), and 10 uM Y-27632 (R&D Systems). Tumors were digested with collagenase solution (5 mL of the above medium with 75 uL collagenase, 124 ug/mL dispase type II, and 0.2% Primocen) for 30 min and then filtered through a 70 um filter (Corning). An organoid pellet was obtained by centrifugation (200x g for 10 min). Organoids were suspended in Matrigel (Corning, Tehama County, CA) with IntestiCult Organoid Growth Medium (#06010, STEMCELL Technologies) and seeded in 12-well plates. Approximately 750 uL of IntestiCult Organoid Growth Medium was added to each well. Organoids were divided into five groups of control, curcumin (3.0 ug/mL), andrographis (30.0 ug/mL), their combination (curcumin; 3.0 ug/mL, andrographis; 30.0 ug/mL), and their combination plus ferrostatin-1 (curcumin; 3.0 ug/mL, andrographis; 30.0 ug/mL; ferrostatin-1; 20 uM). Following forty-eight hours of treatment, the numbers of organoids (<100 um) and their mean sizes were examined using Image J software.

Six 4-week-old male BALB/c nude mice were ordered from the Shanghai Laboratory Animal Center (Shanghai SLAC Laboratory Animal Co., Ltd., China). A total of 5 x 10⁶ TIPE+/+ SW480 cells were suspended in 100 uL of PBS and subcutaneously injected into the right axilla flank of each nude mouse, and the same amount of vector SW480 cells was into the left. At 2 weeks after inoculation, the xenograft tumor size was measured using Vernier calipers every 2 days.

Six 4-week-old male BALB/c nude mice were ordered from the Shanghai Laboratory Animal Center (Shanghai SLAC Laboratory Animal Co., Ltd., China). A total of 5 x 10⁶ TIPE+/+ SW480 cells were suspended in 100 uL of PBS and subcutaneously injected into the right axilla flank of each nude mouse, and the same amount of vector SW480 cells was into the left. At 2 weeks after inoculation, the xenograft tumor size was measured using Vernier calipers every 2 days.

Male BALB/c nude mice (4-6 weeks) were purchased from the Air Force Medical University Laboratory Animal Center. The mice were kept in the SPF environment and had free access to food and water. 3 x 10⁶ SW480 cells were injected subcutaneously into nude mice (n = 4 or 5). Erastin was dissolved in 5% DMSO/corn oil and intraperitoneally injected into nude mice at a dose of 15 mg/kg three times. Three weeks later, mice were anesthetized by intraperitoneal injection of 10% chloral hydrate (35 mg/kg). When mice were successfully anesthetized five minutes later, mice were sacrificed and the tumors were resected and weighed. The tumors were divided into two parts. One sample was lysed and used for protein analysis. The other part was used to test for Ki67 expression.

All nude mice were purchased from Guangdong Medical Laboratory Animal Center. NOS2-overexpressing and control cell lines were transplanted subcutaneously into the bilateral flanks, and appropriate care was given to these animals.

LOVO cells were stably transfected with SRSF9-shRNA1 or NC-shRNA. Caco-2 cells were stably transfected with SRSF9-OE or empty vector. Then the transfected CRC cells (2*105 cells/100 uL) were injected into the right armpit of mouse 6-8-week-old male athymic nude mice. When the tumors reached 50 mm³ at day 7, erastin (40 mg/kg) was administrated to mice by intraperitoneal injection twice every other day.

We obtained the 6-week-old nude mice (BALB/c) from Beijing HFK Bioscience Co., Ltd. and randomly divided into eight groups (n = 5/group): (a) NC + dimethyl sulfoxide (DMSO) group; (b) NC + erastin group; (c) mimic + DMSO group; (d) mimic + erastin group; (e) iNC + DMSO group; (f) iNC + erastin group; (g) inhibitor + DMSO group; (h) inhibitor + erastin group. Control and transfected cells (7 x 10⁶) were subcutaneously injected into the nude mice. After the tumor sizes reached roughly >50 mm³, mice in Groups B, D, F, and H were treated with 15 mg/kg erastin twice every day for about 20 days. Meanwhile, mice in Groups A, C, E, and G were treated with an equal volume DMSO. Besides, tumor formation and mass were monitored and the size of tumor was counted using the formula V = 0.5 x L x W2 (L, length and W, width). At last, after killing the nude mice and then we isolated the mice tumor tissues.

To clarify the role of LINC00239 in vivo, we used 4-week-old male BALB/c nude mice provided by the Experimental Animal Center of the Air Force Military Medical University. HCT116 or SW620 cells (1 x 10⁷ cells) were injected subcutaneously into the right flanks of these mice to establish a CRC xenograft model. One week after the injection of cells, the volume of xenografts was continuously monitored (once a week). Four weeks later, the xenografts were removed, and the weights were measured.

The DLD-1 cell suspension (4 x 10⁶ cells/200 ul) was injected subcutaneously into the right dorsal flank of 5-week-old male BALB/c nude mice (Charles River, China). The mice were randomly divided into four groups (5 mice/group): 1) the control group, 2) the RSL3 group, 3) the cetuximab group, and 4) the RSL3 + cetuximab group. Both RSL3 (5 mg/kg) and cetuximab (13 mg/kg) were administered by intraperitoneal injection in a volume of 100 ul once per day. The tumour volume was calculated as 0.5 x length x width2. After 17 days of treatment, the mice were sacrificed, and the tumours were removed. Then, tumour tissue obtained from the different treated groups was subjected to western blotting and immunohistochemical experiments.

Sixty mice were randomly divided into six groups, (1) the CRC model group (model), (2) mice with RSL3 treatment, (3) mice with Erastin treatment, (4) mice with Ibrutinib treatment, (5) mice with RSL3 and Ibrutinib treatment, and (6) Erastin and Ibrutinib group. Murine subcutaneous tumor model and xenograft tumor mouse model were established and please refer to supplemental method for details. For CRC model group, the mice were treated with PBS for two weeks. For RSL3 group, the mice were intraperitoneal injected with RSL3 (5 mg/kg daily) for two weeks. For Erastin group, the mice were intraperitoneal injected with Erastin (30 mg/kg, twice every other day) for two weeks. For Ibrutinib treatment group, mice were administered in drinking water at a concentration of 0.16 mg/ml for two weeks. Mice were also treated in combination with RSL and Ibrutinib or Erastin and Ibrutinib.

CT26 (1 x 10⁵ cells/100 uL) were injected into thetail veinof male BALB/c mice. Then the mice were randomly divided into saline, vehicle, propofol, and sevoflurane groups (n = 5 per group). Saline, fat emulsion (as vehicle control of propofol), and propofol (200 mg/kg) were intraperitoneally injected, while sevoflurane (1.8-2.0%) was administered by inhalation for 2 h. In another set of experiments, coloncancer cells (CT26 and HT29) were pretreated with two doses of propofol (5 ug/mL, 10 ug/mL) or fat emulsion (as vehicle control of propofol) in a cell culture medium for 2 h. After washing with phosphate-buffered saline (PBS), the cells were harvested,counted on a hemacytometer and prepared. Cells (CT26: 1 x 10⁵ cells/100 uL, HT29: 1 x 10⁶ cells/100 uL) were finnally injected into mice through the tail vein.Lung metastasiswas detected via hematoxylin and eosin staining (HE) or ex vivo bioluminescence imaging.

HCT116-Or or H716 cells (7*10⁶) were suspended into 200 ul matrigel (BD Bioscience), and injected into the subcutaneous tissues of mouse right lower limbs. Animals were grouped randomly (eight mouse per group) and medication was initiated when the average xenograft size was over 100 mm³: Oxaliplatin was given alone weekly through intraperitoneal injection (5 mg/kg), or in combination with liproxstatin-1 through intraperitoneal injection for twice a week (125 mg/kg) and RSL3 through intra-tumor injection (100 mg/kg, in order to achieve better local concentration and reduce the probable systemic toxicity of RSL3) weekly.

To clarify the role of LINC00239 in vivo, we used 4-week-old male BALB/c nude mice provided by the Experimental Animal Center of the Air Force Military Medical University. HCT116 or SW620 cells (1 x 10⁷ cells) were injected subcutaneously into the right flanks of these mice to establish a CRC xenograft model. One week after the injection of cells, the volume of xenografts was continuously monitored (once a week). Four weeks later, the xenografts were removed, and the weights were measured.

HCT116-Or or H716 cells (7*10⁶) were suspended into 200 ul matrigel (BD Bioscience), and injected into the subcutaneous tissues of mouse right lower limbs. Animals were grouped randomly (eight mouse per group) and medication was initiated when the average xenograft size was over 100 mm³: Oxaliplatin was given alone weekly through intraperitoneal injection (5 mg/kg), or in combination with liproxstatin-1 through intraperitoneal injection for twice a week (125 mg/kg) and RSL3 through intra-tumor injection (100 mg/kg, in order to achieve better local concentration and reduce the probable systemic toxicity of RSL3) weekly.

To clarify the role of LINC00239 in vivo, we used 4-week-old male BALB/c nude mice provided by the Experimental Animal Center of the Air Force Military Medical University. HCT116 or SW620 cells (1 x 10⁷ cells) were injected subcutaneously into the right flanks of these mice to establish a CRC xenograft model. One week after the injection of cells, the volume of xenografts was continuously monitored (once a week). Four weeks later, the xenografts were removed, and the weights were measured.

Animals (n = 7) were given 2.5% DSS in drinking water for 5 days and then no treatment for 14 days as one cycle; this process was repeated for three cycles. In the last cycle, 2% DSS water treated to each group and no treatment for 14 days. During the three DSS cycle, 3 mg/kg bromelain were injected daily intraperitoneally and colon and spleen tissues were harvested after three DSS cycle in 57 days to study polyp burden and to perform histological staining.

Animals (n = 7) were given 2.5% DSS in drinking water for 5 days and then no treatment for 14 days as one cycle; this process was repeated for three cycles. In the last cycle, 2% DSS water treated to each group and no treatment for 14 days. During the three DSS cycle, 3 mg/kg bromelain were injected daily intraperitoneally and colon and spleen tissues were harvested after three DSS cycle in 57 days to study polyp burden and to perform histological staining.

Animals (n = 7) were given 2.5% DSS in drinking water for 5 days and then no treatment for 14 days as one cycle; this process was repeated for three cycles. In the last cycle, 2% DSS water treated to each group and no treatment for 14 days. During the three DSS cycle, 3 mg/kg bromelain were injected daily intraperitoneally and colon and spleen tissues were harvested after three DSS cycle in 57 days to study polyp burden and to perform histological staining.

Animals (n = 7) were given 2.5% DSS in drinking water for 5 days and then no treatment for 14 days as one cycle; this process was repeated for three cycles. In the last cycle, 2% DSS water treated to each group and no treatment for 14 days. During the three DSS cycle, 3 mg/kg bromelain were injected daily intraperitoneally and colon and spleen tissues were harvested after three DSS cycle in 57 days to study polyp burden and to perform histological staining.

Animals (n = 7) were given 2.5% DSS in drinking water for 5 days and then no treatment for 14 days as one cycle; this process was repeated for three cycles. In the last cycle, 2% DSS water treated to each group and no treatment for 14 days. During the three DSS cycle, 3 mg/kg bromelain were injected daily intraperitoneally and colon and spleen tissues were harvested after three DSS cycle in 57 days to study polyp burden and to perform histological staining.

Animals (n = 7) were given 2.5% DSS in drinking water for 5 days and then no treatment for 14 days as one cycle; this process was repeated for three cycles. In the last cycle, 2% DSS water treated to each group and no treatment for 14 days. During the three DSS cycle, 3 mg/kg bromelain were injected daily intraperitoneally and colon and spleen tissues were harvested after three DSS cycle in 57 days to study polyp burden and to perform histological staining.

Animals (n = 7) were given 2.5% DSS in drinking water for 5 days and then no treatment for 14 days as one cycle; this process was repeated for three cycles. In the last cycle, 2% DSS water treated to each group and no treatment for 14 days. During the three DSS cycle, 3 mg/kg bromelain were injected daily intraperitoneally and colon and spleen tissues were harvested after three DSS cycle in 57 days to study polyp burden and to perform histological staining.

Animals (n = 7) were given 2.5% DSS in drinking water for 5 days and then no treatment for 14 days as one cycle; this process was repeated for three cycles. In the last cycle, 2% DSS water treated to each group and no treatment for 14 days. During the three DSS cycle, 3 mg/kg bromelain were injected daily intraperitoneally and colon and spleen tissues were harvested after three DSS cycle in 57 days to study polyp burden and to perform histological staining.

The DLD-1 cell suspension (4 x 10⁶ cells/200 ul) was injected subcutaneously into the right dorsal flank of 5-week-old male BALB/c nude mice (Charles River, China). The mice were randomly divided into four groups (5 mice/group): 1) the control group, 2) the RSL3 group, 3) the cetuximab group, and 4) the RSL3 + cetuximab group. Both RSL3 (5 mg/kg) and cetuximab (13 mg/kg) were administered by intraperitoneal injection in a volume of 100 ul once per day. The tumour volume was calculated as 0.5 x length x width2. After 17 days of treatment, the mice were sacrificed, and the tumours were removed. Then, tumour tissue obtained from the different treated groups was subjected to western blotting and immunohistochemical experiments.

Seven-week-old male athymic nude mice (Envigo, Houston, TX) were housed under controlled conditions of light and fed ad libitum. Approximately 5 x 10⁶ parental and 5FUR HCT116 cells were suspended in the matrigel matrix (BD Biosciences, Franklin Lakes, NJ) and subcutaneously injected into mice using a 27-gauge needle (n = 10 per group). Mice were randomly assigned to different treatment groups and 5FU (30 mg/kg body weight) or andrographis (125 mg/kg body weight) or their combination were given intraperitoneally on alternative days for up to 15 days.

CT26 (1 x 10⁵ cells/100 uL) were injected into thetail veinof male BALB/c mice. Then the mice were randomly divided into saline, vehicle, propofol, and sevoflurane groups (n = 5 per group). Saline, fat emulsion (as vehicle control of propofol), and propofol (200 mg/kg) were intraperitoneally injected, while sevoflurane (1.8-2.0%) was administered by inhalation for 2 h. In another set of experiments, coloncancer cells (CT26 and HT29) were pretreated with two doses of propofol (5 ug/mL, 10 ug/mL) or fat emulsion (as vehicle control of propofol) in a cell culture medium for 2 h. After washing with phosphate-buffered saline (PBS), the cells were harvested,counted on a hemacytometer and prepared. Cells (CT26: 1 x 10⁵ cells/100 uL, HT29: 1 x 10⁶ cells/100 uL) were finnally injected into mice through the tail vein.Lung metastasiswas detected via hematoxylin and eosin staining (HE) or ex vivo bioluminescence imaging.

5 x 10⁶ HCT116 cells were inoculated subcutaneously in the underarm of Balb/c nude female mice (5-week old). The inoculated mice were randomly divided into two groups (6 mice each group). When the tumor reached 300 mm³, the drug group was given TalaA intraperitoneally at a dose of 6.0 mg/kg, and the control group was given the same dose of cosolvent-corn oil. The drug (or cosolvent) was injected every 2 days. Body weight and tumor volume were measured every 2 days.

A total 1 x 10⁶ SW620-vector or SW620-FAM98A cells were suspended in 100 ul PBS and injected s.c. into the back of 4- to 6-week-old male BALB/cnude mice (Laboratory Animal Unit, Southern Medical University, China). The sizes of the resulting tumors were measured weekly. Tumor volumes were calculated as follows: total tumor volume (mm³) = (Length x Width2)/2, where Length is the longest length. When the tumor sizes reached about 200 mm³, nude mice in the four groups were given PBS or 5-FU treatment (30 mg/kg, intraperitoneal injection, twice a week), respectively. Nude mice were maintained in a barrier facility in racks filtered with a high-efficiency particulate air filter.

Six-week-old male BALB/c athymic nude mice were purchased from the Experimental Animal Center of Peking (Beijing, China). Stable cells (5 x 10⁶) were seeded into the right flanks of the mice. After the xenografts had grown to 200 mm³, saline as a vehicle or sorafenib (30 mg/kg) was administered by gavage every day, and the mice were euthanized by the cervical dislocation method five weeks later. Before sacrifice, the tumor sizes and body weights were measured twice per week. The tumor volume (V) was calculated as follows: (L x W2)/2 (length, L, and width, W). The xenografts were excised and further assessed.

Six-week-old male C57BL/6J mice were purchased from the Medical Laboratory Animal Center of Guangdong Province (Foshan, China). Forty-five C57BL/6L mice were randomized into 4 groups after 1 week of adaptive feeding: (1) Control group (n = 9); (2) AOM/DSS group (n = 12); (3) AOM/DSS + NaB (orally) group (n = 12); 4) AOM/DSS + NaB (intraperitoneal injection) group (n = 12). The Control group received an intraperitoneal injection of saline solution beginning on day 1, and received sterile drinking water throughout the study. Other three groups received an intraperitoneal injection of 10 mg/kg AOM (Sigma Aldrich) beginning on day 1, and received drinking water containing 2.5% DSS at the second and eighth weeks (2% DSS in the fifth week). Besides, 0.1 M NaB (Sigma Aldrich) was given in drinking water during the whole experiment process in AOM/DSS + NaB (p.o.) group, while AOM/DSS + NaB (i.p.) group was injected intraperitoneally (IP) with 1 g/kg NaB per day.

Five-week-old female BALB/c nude mice were purchased from Beijing Weitong Lihua Experimental Animal Technical Co., Ltd. (Beijing, China). The mice were randomly assigned to the treatment and control groups until the tumor size reached approximately 100 mm³. The mice in the treatment group were injected with 0.174 mg/mL IMCA (100 uL), and those in the control group were injected with an equal volume of normal saline. The nude mice were euthanized, and samples were obtained from their tumor, heart, hepar, kidney, and blood after 33 days of IMCA treatment.

CT26 (1 x 10⁵ cells/100 uL) were injected into thetail veinof male BALB/c mice. Then the mice were randomly divided into saline, vehicle, propofol, and sevoflurane groups (n = 5 per group). Saline, fat emulsion (as vehicle control of propofol), and propofol (200 mg/kg) were intraperitoneally injected, while sevoflurane (1.8-2.0%) was administered by inhalation for 2 h. In another set of experiments, coloncancer cells (CT26 and HT29) were pretreated with two doses of propofol (5 ug/mL, 10 ug/mL) or fat emulsion (as vehicle control of propofol) in a cell culture medium for 2 h. After washing with phosphate-buffered saline (PBS), the cells were harvested,counted on a hemacytometer and prepared. Cells (CT26: 1 x 10⁵ cells/100 uL, HT29: 1 x 10⁶ cells/100 uL) were finnally injected into mice through the tail vein.Lung metastasiswas detected via hematoxylin and eosin staining (HE) or ex vivo bioluminescence imaging.

5 x 10⁶ HCT116 cells were inoculated subcutaneously in the underarm of Balb/c nude female mice (5-week old). The inoculated mice were randomly divided into two groups (6 mice each group). When the tumor reached 300 mm³, the drug group was given TalaA intraperitoneally at a dose of 6.0 mg/kg, and the control group was given the same dose of cosolvent-corn oil. The drug (or cosolvent) was injected every 2 days. Body weight and tumor volume were measured every 2 days.

About 2 x 10⁵ MC38 tumor cells were inoculated subcutaneously into C57BL/6 mice. A total of 100 mg/kg BEBT-908 was intravenously injected every other day for four times initiating from day 4 after tumor cell inoculation. Tumors were harvested on day 12 after inoculation, weighted, mechanically minced and incubated with 50 ug/mL DNase I (Sigma) and 2 mg/mL collagenase P (Sigma) for 20 minutes at 37.

About 2 x 10⁵ MC38 tumor cells were inoculated subcutaneously into C57BL/6 mice. A total of 100 mg/kg BEBT-908 was intravenously injected every other day for four times initiating from day 4 after tumor cell inoculation. Tumors were harvested on day 12 after inoculation, weighted, mechanically minced and incubated with 50 ug/mL DNase I (Sigma) and 2 mg/mL collagenase P (Sigma) for 20 minutes at 37.

The PDX models used in this study were derived from patients (CRC0078 and CRC0121) carrying a quadruple wild-type (KRAS,NRAS,BRAF, andPIK3CA) colorectal tumor. Established tumors (average volume 300 or 500 mm³, as indicated) were treated with the following regimens, either single-agent or in combination: VitC (Sigma, 4 g/kg, intraperitoneal, daily-5 days per week), cetuximab (Merck, 10 mg/kg, intraperitoneal, twice weekly).

The PDX models used in this study were derived from patients (CRC0078 and CRC0121) carrying a quadruple wild-type (KRAS,NRAS,BRAF, andPIK3CA) colorectal tumor. Established tumors (average volume 300 or 500 mm³, as indicated) were treated with the following regimens, either single-agent or in combination: VitC (Sigma, 4 g/kg, intraperitoneal, daily-5 days per week), cetuximab (Merck, 10 mg/kg, intraperitoneal, twice weekly).

5-week-old female BALB/c nude were purchased from Shanghai Slac Laboratory Animal Co., Ltd. HCT116-luc cells were digested and washed by cold PBS for three times, and the final concentration was 2.5 x 10⁶/ml in cold PBS. A volume of 100 ul cell suspension was injected subcutaneously into right dorsal flank of mice. When the tumor tissue in the subcutaneous was macroscopic, the tumor tissues were dissected and embedded into the mesentery of mice.

5-week-old female BALB/c nude were purchased from Shanghai Slac Laboratory Animal Co., Ltd. HCT116-luc cells were digested and washed by cold PBS for three times, and the final concentration was 2.5 x 10⁶/ml in cold PBS. A volume of 100 ul cell suspension was injected subcutaneously into right dorsal flank of mice. When the tumor tissue in the subcutaneous was macroscopic, the tumor tissues were dissected and embedded into the mesentery of mice.

3 x 10⁶ HCT116 cells with stable transfection of lentiviral vectors sh-circ_0007142 or sh-NC were harvested and resuspended in 0.2 mL PBS. Six-week-old male BALB/c nude mice (n = 12) from Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China) were injected with the prepared cell suspension (6 mice/group). During the 4-week observation period, tumour volume (length x width2/2) was detected every week.

3 x 10⁶ HCT116 cells with stable transfection of lentiviral vectors sh-circ_0007142 or sh-NC were harvested and resuspended in 0.2 mL PBS. Six-week-old male BALB/c nude mice (n = 12) from Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China) were injected with the prepared cell suspension (6 mice/group). During the 4-week observation period, tumour volume (length x width2/2) was detected every week.

Six 4-week-old male BALB/c nude mice were ordered from the Shanghai Laboratory Animal Center (Shanghai SLAC Laboratory Animal Co., Ltd., China). A total of 5 x 10⁶ TIPE+/+ SW480 cells were suspended in 100 uL of PBS and subcutaneously injected into the right axilla flank of each nude mouse, and the same amount of vector SW480 cells was into the left. At 2 weeks after inoculation, the xenograft tumor size was measured using Vernier calipers every 2 days.

5-week-old immunodeficient nude mice (male, weight, 16-20 g, n = 40 mice for each group) were purchased from Cyagen bio. Co. (Beijing, China). Before experiments, the mice were adapted to the breeding environment for two weeks. All mice were maintained at a 12 h/12 h light/dark cycle with free access to water and food. A total of 5 x 10⁶ HT-29 or HCT-116 cells were suspended in 100 uL PBS and injected subcutaneously into the right posterior flanks of nude mice. After three weeks, the mice were killed and the tumor xenografts were dissected, weighed and fixed in 10% buffered formaldehyde for further IHC analysis.

CT26 (1 x 10⁵ cells/100 uL) were injected into thetail veinof male BALB/c mice. Then the mice were randomly divided into saline, vehicle, propofol, and sevoflurane groups (n = 5 per group). Saline, fat emulsion (as vehicle control of propofol), and propofol (200 mg/kg) were intraperitoneally injected, while sevoflurane (1.8-2.0%) was administered by inhalation for 2 h. In another set of experiments, coloncancer cells (CT26 and HT29) were pretreated with two doses of propofol (5 ug/mL, 10 ug/mL) or fat emulsion (as vehicle control of propofol) in a cell culture medium for 2 h. After washing with phosphate-buffered saline (PBS), the cells were harvested,counted on a hemacytometer and prepared. Cells (CT26: 1 x 10⁵ cells/100 uL, HT29: 1 x 10⁶ cells/100 uL) were finnally injected into mice through the tail vein.Lung metastasiswas detected via hematoxylin and eosin staining (HE) or ex vivo bioluminescence imaging.

5 x 10⁶ HCT116 cells were inoculated subcutaneously in the underarm of Balb/c nude female mice (5-week old). The inoculated mice were randomly divided into two groups (6 mice each group). When the tumor reached 300 mm³, the drug group was given TalaA intraperitoneally at a dose of 6.0 mg/kg, and the control group was given the same dose of cosolvent-corn oil. The drug (or cosolvent) was injected every 2 days. Body weight and tumor volume were measured every 2 days.

Briefly, surgically resected tumors were maintained in DMEM-F12 (Gibco) supplemented with 1% HEPES (Sigma-Aldrich), 1% L-glutamine (Gibco), 10% FBS (Gibco), 2% penicillin/streptomycin (Sigma-Aldrich), and 10 uM Y-27632 (R&D Systems). Tumors were digested with collagenase solution (5 mL of the above medium with 75 uL collagenase, 124 ug/mL dispase type II, and 0.2% Primocen) for 30 min and then filtered through a 70 um filter (Corning). An organoid pellet was obtained by centrifugation (200x g for 10 min). Organoids were suspended in Matrigel (Corning, Tehama County, CA) with IntestiCult Organoid Growth Medium (#06010, STEMCELL Technologies) and seeded in 12-well plates. Approximately 750 uL of IntestiCult Organoid Growth Medium was added to each well. Organoids were divided into five groups of control, curcumin (3.0 ug/mL), andrographis (30.0 ug/mL), their combination (curcumin; 3.0 ug/mL, andrographis; 30.0 ug/mL), and their combination plus ferrostatin-1 (curcumin; 3.0 ug/mL, andrographis; 30.0 ug/mL; ferrostatin-1; 20 uM). Following forty-eight hours of treatment, the numbers of organoids (<100 um) and their mean sizes were examined using Image J software.

Briefly, surgically resected tumors were maintained in DMEM-F12 (Gibco) supplemented with 1% HEPES (Sigma-Aldrich), 1% L-glutamine (Gibco), 10% FBS (Gibco), 2% penicillin/streptomycin (Sigma-Aldrich), and 10 uM Y-27632 (R&D Systems). Tumors were digested with collagenase solution (5 mL of the above medium with 75 uL collagenase, 124 ug/mL dispase type II, and 0.2% Primocen) for 30 min and then filtered through a 70 um filter (Corning). An organoid pellet was obtained by centrifugation (200x g for 10 min). Organoids were suspended in Matrigel (Corning, Tehama County, CA) with IntestiCult Organoid Growth Medium (#06010, STEMCELL Technologies) and seeded in 12-well plates. Approximately 750 uL of IntestiCult Organoid Growth Medium was added to each well. Organoids were divided into five groups of control, curcumin (3.0 ug/mL), andrographis (30.0 ug/mL), their combination (curcumin; 3.0 ug/mL, andrographis; 30.0 ug/mL), and their combination plus ferrostatin-1 (curcumin; 3.0 ug/mL, andrographis; 30.0 ug/mL; ferrostatin-1; 20 uM). Following forty-eight hours of treatment, the numbers of organoids (<100 um) and their mean sizes were examined using Image J software.