Cell lines and culture conditions

The major cell lines used in the cancer study were as follows. The HCT116 cell line was purchased from ATCC and cultured in McCoy’s 5A medium supplemented with 10% fetal bovine serum (FBS; GeminiBio, West Sacramento, CA) and 1% penicillin/streptomycin (pen/strep; Corning Inc., Glendale, AZ). The human triple-negative breast cancer cell line MDA-MB-231 and its derivative MDA-MB-231-GFP/Luc have been previously described [68] and were cultured in Dulbecco’s modified Eagle medium (DMEM, Corning Inc., Glendale, AZ) supplemented with 10% FBS and 1% pen/strep. The isogenic Cetuximab-sensitive LIM1215 (KRAS wildtype) and Cetuximab-resistant LIM1215R4 (KRAS mutant) colorectal cancer cell lines have been previously described [69] and were cultured in RPMI-1640 media (Corning Inc., Corning, NY), supplemented with 10% FBS and 1% pen/strep. Mouse embryonic fibroblast cell line (MEF), mouse acinar pancreatic cancer cell line 266–6 (Gift from Dr. Hamid M. Said, UCI) were cultured in DMEM supplemented with 10% FBS and 1% pen/strep. Human head and neck cancer cells SCC15, SCC25, and SCC351 (Gift from Dr. Vicky Yamamoto, USC) have been previously described [12] and were cultured in DMEM supplemented with 10% FBS and 1% pen/strep. The cell lines used in the survey for the prevalence of Na+/K+-ATPase α1 and α3 (A427, A549, H522, RKO, SW480, PanC1, CFPAC1, MCF7, C4-2B, SK-MEL-28, H929, and HepG2) and their culture conditions were previously described [20, 70,71,72].

The cell lines used in the virus study were as follows. The African green monkey kidney epithelial cell line Vero E6 expressing ACE2 (Vero E6-ACE2) was a gift from Dr. Younho Choi (University of Southern California). The human colorectal adenocarcinoma cell line Caco-2 expressing ACE2 (Caco-2-ACE2) was a gift from Drs. GuanQun Liu and Michaela Gack (Florida Research and Innovation Center). Human Lung Carcinoma Cells A549 Expressing Human Angiotensin-Converting Enzyme 2 (A549-ACE2) was obtained from the Centers for Disease Control and Prevention and obtained through BEI Resources, NIAID, NIH (NR-53821). Vero E6-ACE2 cells were cultured in DMEM supplemented with 10% FBS, 1% pen/strep and 1 µg/ml puromycin. A549-ACE2 cells were cultured in DMEM supplemented with 10% FBS, 1% pen/strep and 100 µg/ml blasticidin. All cells were maintained at 370C in a humidified atmosphere of 5% CO2 and 95% air.

The cell lines were routinely tested for mycoplasma contamination.

Compounds and treatment conditions

Lanatoside C, digoxin, bufalin, ouabain, oleandrin, chloroquine, and 3-methyladenine were purchased from Millipore Sigma (St. Louis, MO). The proteasome inhibitors MG101, MG115, and MG132 were purchased from Selleckchem (Houston, TX). The ER-stress inducers thapsigargin (Tg), tunicamycin (Tu), and 2-deoxy-D-glucose (2-DG), and the autophagy inhibitor bafilomycin A1 were purchased from Cayman Chemical (Ann Arbor, MI). Remdesivir (HY-104077) and Nirmatrelvir (HY-138687) were purchased from MedChemExpress (Monmouth Junction, NJ). All drugs were dissolved in DMSO except for 3-MA and 2-DG which were dissolved in sterile double distilled water. The final concentration of DMSO in cell culture were either 0.1% or 1%. To induce ER stress, the cells were treated with Tg at 300 nM, Tu at 1.5 μg/ml, or 2-DG at 10 mM for 24 h. In combination treatment, cardiac glycosides and the ER-stress inducers were added to the cells at the same time and incubated for 24 h. For proteasome and autophagy inhibition, cells were treated with oleandrin, Tg, and the proteasome inhibitors MG101 (10 μM), MG115 (10 μM), MG132 (10 μM) or autophagy inhibitors 3-MA (10 mM), chloroquine (20μM), and bafilomycin A1 (100 nM) at the same time and incubated for 24 h. For the drug-OLN combinational treatment study, 15 nM of OLN was treated alone or in combination with Remdesivir or Nirmatrelvir at concentrations of 100 nM and 500 nM, respectively. For all experiments, DMSO was used as vehicle control.

Hypoxia induction and glucose starvation

For hypoxia induction, we utilized the Galaxy 48r Incubator CO2/O2/N2 (Eppendorf, Hamburg Germany). The chamber was set at 0.1% O2 and 5% CO2. Regular culture medium (DMEM 10% FBS 1% pen/strep) was incubated at 0.1% O2 and 5% CO2 for 48 h before the start of the experiment to decrease the oxygen concentration in the medium. This is the “equilibrated medium”. HCT116 and MDA-MB-231 cells were seed at 5 × 105 cells per 6 cm dish and allowed to attached overnight. Next day, the cells were switched to the “equilibrated medium” prepared above and treated with DMSO vehicle control or 35 nM of oleandrin and incubated at 0.1% O2 and 5% CO2 for 24 h. An identical set of cells were incubated at regular culture condition (20% O2 and 5% CO2). After 24-h incubation period, cells were washed once with ice cold PBS and immediately lysed with RIPA buffer. Cell lysates were subjected to Western blot analysis to detect the proteins of interest.

For glucose starvation, HCT116 and MDA-MB-231 were seeded at 5 × 105 cells per 6 cm dish and allowed to attach overnight. Next day, cells were washed twice with PBS to remove any trace of glucose and incubated in glucose-free DMEM supplemented with 1% pen/strep (Thermo Fisher Scientific, Cat# 11966025, Waltham, MA) and treated with DMSO vehicle control or 35 nM of oleandrin for 24 h. Cells were then washed with PBS and lysed with RIPA buffer. Cell lysates were subjected to Western blot analysis to detect the proteins of interest.

Antibodies for immunoblots

The following antibodies were used in this study. Primary antibodies: mouse anti-GRP78 antibody (1:1000, BD Biosciences, San Jose, CA, 610979), rat anti-GRP94 antibody (1:1000, Enzo Life Sciences, Farmingdale, NY, SPA-851), mouse anti-HSP70 antibody (1:1000, Santa Cruz Biotechnology, Inc., Dallas, TX, sc-66048), rabbit anti-calnexin antibody (1:2000, Enzo Life Sciences, ADI-SPA-860), rabbit anti-PDI antibody (1:2000, Enzo Life Sciences, ADI-SPA-890), mouse anti-β-actin antibody (1:5000, ProteinTech, Rosemont, IL, 66009–1-Ig), mouse anti-FLAG M2 antibody (1:2000, MilliporeSigma, F1804), rabbit anti-cleaved PARP (Asp214) antibody (1:1000, Cell Signaling, Danvers, MA, #5625), rabbit anti-cleaved caspase 3 (Asp175) antibody (1:1000, Cell Signaling, #9661), rabbit anti-cleaved caspase 7 (Asp198) antibody (1:1000, Cell Signaling, #8438), mouse anti-E-cadherin antibody (1:1000, BD Biosciences, 610181), mouse anti-GAPDH antibody (1:5000, Santa Cruz Biotechnology, Inc., sc-32233), rabbit anti-Sodium/Potassium ATPase α3 antibody (1:1000, GeneTex, Inc., Irvine, CA, GTX53511), rabbit anti-Na,K-ATPase α1 (1:1000, Cell Signaling, #3010), mouse anti-Puromycin antibody (1:1000, Development Studies Hybridoma Bank, Iowa City, IA, PMY-2A4), rabbit anti-ATF4 (CREB2) antibody (1:500, Santa Cruz Biotechnology, Inc., sc-200), mouse anti-CHOP (L63F7) antibody (1:1000, Cell Signaling, #2895), rabbit anti-Phospho-eIF2α (Ser51) antibody (1:1000, Cell Signaling, #9721), mouse anti-ATF6 antibody (1:1000, IMGENEX, San Diego, CA), rabbit anti-ACE2 (1:1000; Proteintech, Rosemont, IL, 21,115–1-P), mouse-anti-Annexin A2 (1:1000, BD Biosciences, 610068). Secondary antibodies: horseradish peroxidase (HRP) conjugated goat anti-mouse (sc-2005), goat anti-rabbit (sc-2004), and goat anti-rat (sc-2006) antibodies (1:1000, Santa Cruz Biotechnology, Inc.), mouse IgG \(\kappa\) binding protein conjugated to HRP (1:1000, Santa Cruz Biotechnology, Inc., sc-516102), mouse anti-rabbit IgG conjugated to HRP (1:1000, Santa Cruz Biotechnology, Inc., sc-2357), goat anti-mouse IRDye® 800CW (1:1000, LI-COR Biosciences), goat anti-rabbit IRDye® 680RD (1:1000, LI-COR Biosciences).

Plasmids

The construction of the FLAG-GRP78 expression vector has been described previously [15]. The pcDNA3 empty vector was used as control.

Viruses

The following viral stocks were deposited by the Centers for Disease Control and Prevention and obtained through BEI Resources, NIAID, NIH: SARS-Related Coronavirus 2, Isolate USA-WA1/2020 (Original Strain) (NR-52281), SARS-Related Coronavirus 2, Isolate hCoV-19/USA/MD-HP20874/2021 (Omicron Variant, Strain B.1.1.529) (NR-56461), SARS-Related Coronavirus 2, Isolate hCoV-19/South Africa/KRISP-K005325/2020 (Beta Variant) (NR-54009) and SARS-Related Coronavirus 2, Isolate hCoV-19/USA/MD-HP05285/2021 (Delta Variant) (NR-55671).

All the viruses used in this study were propagated in Caco-2-ACE2 cells in DMEM incubated at 37 °C with 5% CO2 for 48 h. Supernatant was collected, passed through a 0.45 μm pore size polyethersulfone (PES) syringe filter, aliquoted and stored at -80ºC until further use. The virus titer was determined by plaque assay as previously described [73].

Transfection of plasmids and siRNAs

Expression vectors were transfected into HCT116 cells using the BioT Transfection Reagent (Bioland Scientific, Paramount, CA) according to manufacturer’s recommendations. The cells were incubated with the transfection mix for 24 h to allow for protein expression before the addition of Tg and OLN and were incubated for a further 24 h before harvesting for immunoblot analysis.

The control siRNA (siCtrl) and siRNA targeting the Na+/K+-ATPase α3 isoform were purchased from Integrated DNA Technologies (IDT, Coralville, IA) with proprietary sequences. The siRNAs were transfected into cells using Lipofectamine™ RNAiMAX Transfection Reagent (ThermoFisher, Waltham, MA) according to manufacturer’s recommendations. The cells were incubated with the transfection mix for 24 h to allow for gene knockdown before the addition of Tg and OLN and were incubated for a further 24 h before harvesting for immunoblot analysis.

Transfection of plasmids for viral studies, virus infection and cell harvest

Vero E6-ACE2 cells were seeded in 6-well plate (106 cells/well) and cultured in DMEM supplemented with 10% FBS and 1% pen/strep and incubated at 37 °C overnight. The cells were washed once with fresh DMEM and transfected with 2 μg of either pcDNA3 empty vector or FLAG-GRP78 using BioT transfection reagent (Bioland Scientific) according to the manufacturer’s instructions. At 48 h post-transfection, the cells were washed once with fresh DMEM and infected with 0.01 MOI of virus in each well. The plates were incubated on a rocker at 37 °C for 45 min for virus adsorption. The virus inoculum was then removed and replaced by fresh DMEM containing twofold serial dilutions of OLN and placed in 37 °C incubator for 3 days. The supernatant was collected, filtered with 0.45 μm syringe filter and stored at -80ºC. The supernatant samples were subjected to plaque assay for virus titration.

A549-ACE2 cells were seeded in 6-well plates and incubated overnight. Next day, the cells were washed once with fresh DMEM and infected with 0.5 MOI of virus in each well. The plates were incubated on a rocker at 37 °C for 45 min for virus adsorption. The virus inoculum was then removed and replaced by fresh DMEM containing the indicated concentrations of OLN and placed in a 37 °C CO2 incubator. The cell pellets were collected and stored at − 80 °C. The cell pellet samples were lysed, and cell lysates were subjected to immunoblot analysis as described above.

RT-qPCR

Cells in 6 cm culture dishes were washed 3 times with ice cold DPBS and scrapped with 1 ml of TRI Reagent (Millipore-Sigma). The total RNA was extracted according to manufacturer’s instructions. Total RNA was measured by a NanoDrop 1000 Machine (ThermoFisher, Waltham, MA) to determine the concentration and purity. To synthesize complementary DNA (cDNA), 1 μg of total RNA was used in qScript cDNA SuperMix First-Strand cDNA Synthesis Kit (QuantaBio, Beverly, MA) according to manufacturer’s recommendations. For RT-qPCR analysis, cDNA was amplified by the KAPA SYBR® FAST qPCR Master Mix (Roche Sequencing and Life Science, Wilmington, MA) and detected by the Stratagene MX3000P Real-Time QPCR System (Agilent, Santa Clara, CA) with the following PCR conditions (40 cycles, 15 s at 950C, 15 s at 550C, 30 s at 720C). Melting curve analysis was performed to ascertain the specificity of the primers. One single peak was observed for each PCR product. The primers for human GRP78 are 5’-GGTGAAAGACCCCTGACAAA-3’ and 5’-GTCAGGCGATTCTGGTCATT-3’, for human β-actin are 5’-TCCCTGGAGAAGAGCTACGA-3’ and 5’-AGCACTGTGTTGGCGTACAG-3’.

For XBP-1 splicing assay, 1 μl of cDNA was amplified in regular PCR with the following conditions (35 cycles, 950C for 30 s, 580C for 30 s, 720C for 45 s). The primers for human XBP-1 are Forward: 5’-TTACGAGAAAACTCATGGC-3’ and Reverse: 5’- GGGTCCAAGTTGTCCAGAATGC-3’. PCR products were electrophoresed on 3% agarose gel and DNA bands were visualized by the ChemiDoc XRS + Imager (Bio-Rad Laboratories, Hercules, CA).

Cell surface biotinylation and purification

Cells were seeded on 6 cm culture dishes and treated as indicated. After 24 h drug treatment, cells were washed 3 times with ice cold PBS and incubated with EZ-Link Sulfo-NHS-SS-Biotin (Thermo Scientific, Waltham, MA) dissolved in PBS at a concentration of 0.5 mg/ml for 30 min at 40C with gentle agitation. The biotinylation labeling solution was removed after 30 min and the reaction was quenched by Tris–Cl buffer pH 7.4 in cold PBS. Cells were then washed 3 times with PBS and lysed with RIPA buffer supplemented with Protease and Phosphatase inhibitor (ThermoFisher, Waltham, MA). Part of the lysate was saved as whole cell lysate to measure the total level of the indicated proteins. The remaining lysate was incubated with High Capacity NeutrAvidin Agarose Beads (ThermoFisher, Waltham, MA) for 1 h at room temperature to purify the biotinylated cell surface proteins. After 1 h, the unbound fraction was removed, and the agarose beads were wash 10 times with RIPA buffer. The biotinylated cell surface proteins bound to the beads were released by addition of 50 μl of 2X SDS-PAGE sample buffer with heating at 950C for 5 min and analyzed by Western blot.

Immunoblot analysis

Whole cell lysates were prepared by scrapping cells from 6 or 10 cm cell culture dishes with ice cold RIPA buffer (50 nM Tris–HCl, 150 nM NaCl, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% Sodium dodecyl sulfate) supplemental with Protease and Phosphatase inhibitor cocktail (ThermoFisher, Waltham, MA). The crude lysates were incubated on ice for 30 min followed by centrifugation at 13,000 RPM at 40C for 15 min. The clarified lysate solution containing soluble proteins was transferred to a new tube and mixed with 6X SDS sample buffer (0.375 M Tris pH 6.8, 12% SDS, 60% Glycerol, 0.6 M β-mercaptoethanol, 0.06% bromophenol blue). The protein samples were then heated at 950C for 5 min to denature all proteins. Protein samples were electrophoresed through 8%, 10%, or 12% SDS-PAGE gels and transferred to supported nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA). Immobilized proteins on the membrane were blocked with 5% non-fat dry milk in TBST solution and incubated with primary antibodies either for 2 h at room temperature or overnight at 40C followed by 2 h incubation in secondary antibodies at room temperature. Protein bands were developed by enhanced luminol-based chemiluminescent substrates (ThermoFisher, Waltham, MA) and detected by the ChemiDoc XRS + Imager (Bio-Rad Laboratories, Hercules, CA) or the LI-COR Odyssey 9120 Imaging System (LI-COR Biosciences, Lincoln, NE). Image analysis and quantitation of the band intensity were performed using the Image Lab Software Version 4.0.1 build 6 (Bio-Rad Laboratories) or Odyssey V3.0 Image Analysis software (LI-COR Biosciences).

Immunofluorescence

Cells were seeded on Millicell EZ SLIDE (MilliporeSigma, PEZGS0816) and allowed to attach overnight. Next day, the cells were treated with drugs as indicated for 24 h. Then the media was removed, and the cells were washed with PBS three times and fixed with 4% paraformaldehyde (PFA) and permeabilized in 0.02% Triton X-100 for 10 min at room temperature. For staining of cell surface GRP78, the cells were fixed with 4% PFA but not permeabilized. After incubation with blocking buffer (5% BSA, 0.1% Tween-20, PBS) for 1 h, cells were incubated at 4 °C overnight with primary antibodies diluted in PBST in a humidified chamber at 4 °C. Primary antibody: mouse anti-GRP78 (1:500, MAb159, Gift from Dr. Parkash Gill, USC). Cells were washed three times with PBS and were incubated with Alexa Fluor-conjugated secondary antibodies for 30 min at room temperature, followed by three more washes with PBS. Secondary antibody: Alexa Fluor 488 goat anti-mouse antibody (1:500, Thermo-Fisher Scientific, #A-11001). Cells were mounted with VECTASHIELD Antifade Mounting Medium with DAPI (Vector Laboratories, Inc., #H1200). Cell images were acquired with Leica SP8 LIGHTNING Confocal Microscope using a 63 × oil objective.

Immunohistochemistry

Immunostaining of paraffin-embedded tumor tissue sections was performed as described previously [74]. Tumor tissue sections were incubated at 4 °C overnight with primary antibodies. The antibodies used were: GRP78 (1:500, Abcam, Cambridge, MA, #ab108613). The immunostaining was visualized using the SignalStain Boost IHC Detection Reagent (HRP, Rabbit) (Cell Signaling, Danvers, MA, #8114) as per manufacturer’s protocol.

Analysis of polysomal-associated GRP78 mRNA

Sucrose solution was prepared in polysome extraction buffer (10 mM HEPES, 100 mM KCl, 5 mM MgCl2, pH 7.4, 100 μg/ml cycloheximide, 5 mM DTT). Sucrose gradients were prepared in SW41 ultracentrifuge tubes by mixing 15% and 45% sucrose solutions using a BioComp Gradient Master 108, according to the manufacturer’s instructions. Cells were lysed in polysome extraction buffer with added 1% Triton-X and RNAase out. Equal amounts of supernatants were loaded on top of the gradients and then centrifuged at 37,000 RPM at 4 °C for 2 h in an SW41 rotor of an Optima XPN 80 ultracentrifuge. Gradients were fractionated with a speed of 800 μl/min using a Biocomp piston gradient fractionator, which recorded the OD254nm. Fractions corresponding to 60 s intervals were collected and RNA was isolated from individual fractions using TRIzol LS Reagent (no. 10296010, Invitrogen), spiked with Firefly Luciferase (FLuc) control RNA (no L4561, Promega) as internal control. FLuc was diluted in TRIzol LS before addition to all fractions in a concentration of 0.001 μg/ml per sample. RNA quality was checked by NanoDrop and running of RNA in 1% agarose gels. These gels also show ribosomal RNA to determine monosome fractions. A fixed volume of RNA (5 μl) from each fraction was reverse transcribed to cDNA using a cDNA Reverse Transcription kit (no. 4387406, Thermo Fisher Scientific). cDNA samples were diluted 1:40, and 2 μl of template was used for qPCR (40 cycles: 15 s at 95 °C, 1 min at 60 °C) with Applied Biosystems TaqMan Assays. The probe used for GRP78/HSPA5 is Hs00946350_g1 (Catalogue #4351372) and for luciferase Mr03987587-mr (Catalogue #4331182). RNA levels were quantified, normalized to internal FLuc as control, summed across all fractions, analyzed, and presented as percentages of this total.

Puromycin labeling assay

HEK293T cells were seeded on 6 cm dishes and allowed to attached overnight. Next day, the cells were treated with OLN (0–100 nM) alone or in combination with Tg (300 nM) for 8 h followed by labeling with puromycin (10 μg/ml) for 30 min. Cells were then lysed and whole cell lysates were subjected to Western blot analysis for puromycin-labeled proteins.

Cell viability assay

Vero E6-ACE2 cells or A549-ACE2 cells were seeded at a density of 1 × 104 cells per well in a 96-well flat bottom plate with DMEM supplemented with 10% FBS and 1% pen/strep. After overnight culture in 37 °C incubator, the medium was removed and replaced with serum-free DMEM and indicated concentrations of OLN at a final DMSO concentration of 0.1%. As a non-treatment control, 0.1% DMSO was used throughout the assay. Cell viability was measured at 48 and 72 h post-treatment using the (4-[3-(4-Iodophenyl)-2-(4-nitro-phenyl)-2H-5-tetrazolio]-1,3-benzene sulfonate) WST-1 cell proliferation assay kit (Takara Bio USA, Inc., San Jose, CA) according to the manufacturer’s recommendation. Colorimetric quantitation was achieved using a Model 680 Microplate Reader (Bio-Rad Laboratories, Hercules, CA) at a wavelength of 450 nm and subtracted by a reference wavelength of 650 nm. The background absorbance of blank media (DMEM 1% pen/strep) was also measured and subtracted from the sample reading.

Colony formation assay

HCT116 cells were seeded at a density of 1 × 103 cells per well in 6-well plates. Following drugs treatment for 24 h, the media was replaced with fresh media and the cells were allowed to grow for 2 weeks with frequent media change every 3 days. After the 2-week period, cells were washed with PBS, fixed in 100% methanol for 15 min at room temperature followed by Coomassie Blue staining for 30 min to visualize surviving colonies. The number of surviving colonies were counted by ImageJ software (U.S. National Institutes of Health, Bethesda, MD).

Plaque reduction assay

Evaluating the antiviral activities of OLN, Remdesivir and Nirmatrelvir was done by plaque reduction assay as described previously with minor modifications [75]. Briefly, confluent monolayers of Vero E6-ACE2 cells in 6-well plates were washed once with DMEM and infected with approximately 30 to 100 plaque forming units (PFUs) of virus in each well. Virus-free DMEM was used for mock infections. The plates were incubated on a rocker at 37 °C for 45 min for virus adsorption. The virus inoculum was then removed and replaced by overlay media (DMEM containing 1% low-melting agarose without serum) containing desired concentrations of either OLN, Remdesivir or Nirmatrelvir and placed in 5% CO2 incubator at 37 °C until plaques could be visualized under light. The cells were fixed with 4% formaldehyde solution for at least 30 min and the overlaid agarose was removed and stained with 0.2% (w/v) crystal violet solution. The plaques were counted by visual examination and the size of the plaques were measured by scale loupe.

Patient-derived colon cancer organoids culture

For organoid generation, metastatic colon tumor tissue localized to liver was received from a consented patient following Institutional Review Board (IRB) approval at the Norris Comprehensive Cancer Center of the University of Southern California, Los Angeles, California. Patient‐derived metastatic colorectal tumor organoids (PDOs) were developed as previously described [63]. In brief, tissue was minced, digested with collagenase (Millipore, Temecula, CA), hyaluronidase (MP Biomedicals, Solon, OH) and L-Y27632 (Sigma, St. Louis, MO), cells were passed through a 100 μm strainer, washed with media, resuspended in BME (R&D System, Minneapolis, MN) and cultured with colon tumor organoid (CTO) media: ADMEM/F12 (ThermoFisher Scientific, Waltham, MA), 10% FBS (Gemini Bio, Sacramento, CA, USA), 1% P/S (Gemini Bio, Sacramento, CA), 1 × GlutaMax (ThermoFisher Scientific, Waltham, MA), 1 × HEPES (ThermoFisher Scientific), 1 × B-27 (ThermoFisher Scientific), 100 ng/ml noggin (Tonbo Biosciences, San Diego, CA), 1 × N-2 (ThermoFisher Scientific), 10 mM nicotinamide (Sigma-Aldrich), 1 mM N-acetylcysteine (Sigma-Aldrich), 500 nM A-83–01 (MilliporeSigma, Burlington, MA), 50 ng/ml EGF (Life Technologies, Grand Island, NY), and 10 μM SB202190 (Sigma-Aldrich).

OLN drug response curve and cell harvest

For cell line-based drug treatment studies, LIM1215 WT and R4 cells were seeded at 1.5 × 103 cells per well in 96-well black-wall, clear-bottom plates (Revvity, Waltham, MA). The following day, cells were dosed with OLN at concentrations ranging from 1 to 500 nM. After 3 days of OLN treatment, cells were stained with 5 μg/ml Hoechst 33342 (Life Technologies, Grand Island, NY) and 5 µg/ml propidium iodide (Life Technologies) prior to imaging to identify live/dead cells on the Operetta High Content Screening (HCS) system (Revvity, Waltham, MA). Cells were then segmented based on Hoechst-stained nuclei and classified as alive or dead based on propidium iodide intensity using Harmony software (Revvity, Waltham, WA) [76].

For PDO-based drug treatment studies, PDOs were fragmented to single cells with TrypLE Express (ThermoFisher Scientific) and filtered through a 40 μm strainer to remove aggregates, seeded on a 96-well white-wall, clear-bottom plate (Corning, Corning, NY) at a concentration of 2.5 × 103 cells per well, and topped with 100 μl of CTO media. PDOs were incubated for 4 days and then treated with OLN at different concentrations from 5 to 400 nM for 3 days at normoxic (21% oxygen) and hypoxic (1% oxygen) conditions (O2 level maintained using BioSpherix hypoxia chamber glovebox and incubator). On day 3 of treatment, 100 μl of CellTiter-Glo 3D solution (Promega, WI) was added to each well, and cell viability was analyzed by measuring luminescence with a BioTek Synergy Neo2 Multi-Mode Reader (Agilent Technologies, Santa Clara, CA). Images of PDOs were also acquired after 3 days of treatment using the Operetta CLS HCS (Revvity, Waltham, WA) and displayed with maximum projections of 24 z-stacks ranging from 10–470 μm in increments of 20 μm.

LIM1215 WT and R4 cells cultured in 10 cm dishes were treated with OLN at different concentrations from 5 to 50 nM for 3 days before being harvested, centrifuged, and stored as snap frozen pellets. PDOs were cultured in normoxia (21% oxygen) and hypoxia (1% oxygen) conditions for 3 days before being harvested, removed from BME with Gentle Cell Dissociation Reagent (StemCell Technologies, Cambridge, MA), centrifuged, and stored as snap frozen pellets. Cell pellets were subjected to immunoblot analysis as mentioned above.

Tumor xenograft model

1 × 106 MDA-MB-231-GFP/Luc cells were orthotopically injected into the #4 fat pad of 6- to 8-week-old female NSG mice (5 mice in each group) (Jackson Laboratory, stock #005557). One week after implantation of tumor, the mice were treated with vehicle control or OLN at a concentration of 0.3 mg/kg by I.P. injection every other day excluding weekend for a 2-week period. At the end of the treatment period, the tumors were collected for Western blot and immunohistochemical analysis.

TCGA analysis

GRP78 gene expression analysis in human normal colon and breast and colorectal adenocarcinoma and breast cancer tissues was obtained from the Gene Expression Profiling Interactive Analysis 2 (GEPIA2) [77]. The “normal tissues” included the matched adjacent normal tissues from the same patients in the TCGA database in addition to normal tissue samples from unrelated donors from the Genotype-Tissue Expression (GTEx) database [78].

Statistical analysis

All pairwise comparisons were analyzed by unpaired 2-tailed Student’s t test using Microsoft Excel. All graphs were produced with GraphPad Prism version 10.0 (GraphPad Software, San Diego, CA). Data were presented as means ± Standard Deviation (S.D.). A p-value of ≤ 0.05 is signified by *, p-value of ≤ 0.01 by **, p-value of ≤ 0.001 by ***, and p-value of ≤ 0.0001 by ****, n.s. denotes not significant.