Abstract # 52

Understanding Cetuximab Response andImmune Modulation in Colorectal Cancer using PatientDerived Organotypic Tumor Spheroids
Ketki Bhisea, Emma Cuia, Kirti Khardenavisa, Anthony Attardoa, Danielle Hageea, Sinal Patela, Julia Vaila, Amiraaa,baa
Aref , Nicholas Barris , Aaron Goldman , Alyssa Martin , Michael A. Perricone *aXsphera Biosciences Inc., 6 Tide St., Boston, MA 02210; bDepartment of Medicine, Harvard Medical School, Boston, Massachusetts 02115, United States; *Corresponding author

BackgroundMolecular pathways of tumor suppressionNoteworthy findings:

Cetuximab, an epidermal growth factor receptor (EGFR) specificAmong CRC samples derived from KRASwt tumors, 6 out of 16

monoclonal IgG1 antibody, holds considerable importance in the(37.5%) exhibited a cytotoxic response to Cetuximab comparedtreatment against Kirsten Rat Sarcoma virus (KRAS) wild typeto the IgG control, slightly higher than the ~21% response rate(KRASwt)

observed in CRC patients.

(CRC).

Cetuximab

triggersthe•No KRASmut samples tested responded to Cetuximab treatment.

downstream signaling pathways including IL6/STAT3, PI3K/AKT,•

There was evidence of rapid initiation of ADCC in CRC PDOTS

internalizationofEGFR,

whichinturnsuppresses

samples responsive to Cetuximab.

MTORC1. KRAS mutations are associated with resistance to•Cetuximab treatment in CRC, thus anti-EGFR therapies are only

Among treatment-responsive CRC samples, suppression ofapproved for KRASwt. Cytotoxic response by Cetuximab is also

pathways downstream of EGFR signaling was evident at day 3known to be mediated by antibody-dependent cellular cytotoxicity

post-treatment.
•(ADCC). Here, an ex vivo patient-derived organotypic tumor

Data generated from the PDOTS platform are consistent withspheroids (PDOTS) platform1 provides insight into the response,

clinical findings2, indicating the platform’s effectiveness inmechanistic pathways and resistance dynamics to Cetuximab

maintaining patients’ tumor microenvironment and unravelingtreatment in PDOTS and supports a better understanding of thethe complexity of tumor-targeted therapies.

Sample Characterization
Pathology

Patientsample

Mechanical&enzymaticdigestion

Mutation analysis>100 µmTumor profile (flow cytometry)

Delta Area Weighted % DeadDay 3KRAS

Our patented ex vivo microfluidic platform

PDOTS (i.e tumor spheroids)
recapitulate patients’ tumor
microenvironment, including
infiltrated immune cells

expression

profilescompared

between

Cetuximab-responsive and non-responsive PDOTS. Treatmentresponsivegroup(inred)showedevidence

oftumorsuppression by downregulation of genes responsible for

MTORC1 Signaling

PI3K-Akt

Statics
KRASWT

The platform conserves native tumor milieu

Genehypoxia, compared to the non-responsive group (in blue).

<40 µm

PDOTS
40 µm~100 µm

5:Responsive

formation of mitotic spindle, glycolysis, metabolic stress andResponse threshold

Non-responsive

Loaded into microfluidic
devices, followed by drug
treatment.
Post-treatment analytics
include:End-point cytotoxicity
Cytokines
Gene expression

Hypoxia

Non-responsive

Fig.

Results
Cetuximab cytotoxic response on day 3

variability in patient response.

Metabolic Stress

Delta Norm CountcancerGlycolysis

Responsive

KRASmut

Fig. 2: Cetuximab cytotoxic response on day 3, expressed as area

Delta Norm Count

colorectalMitotic Spindle

Delta Norm Count•weighted (AW) % dead. AW % Dead is calculated by dividing thesum of dead area of all spheroids in a given channel by the sum ofNon-responsive

total area of all spheroids. With n=32 treated with 300 µg/mL

Responsive

Cetuximab, 18.75% showed significant response (p<0.2). Samples

Fig. 6: Gene expression profiles for specific pathways between

were categorized into treatment-responsive (R) and non-responsive

Cetuximab-responsive and non-responsive PDOTS. Pathways(NR) groups. All responsive samples were KRASWT.

downstream of EGFR signaling, PI3K-Akt and MTORC1signaling, were suppressed in the treatment-responsive group

Cytokine profiles

Brightfield
TNFαIL-6IL-1Ra

IL-1β(red), compared to the non-responsive group (in blue).

Differentially expressed genes inCetuximab-responsive CRC PDOTS
Spheroid channel

Media/drug channels

Hoechst-Propidium iodide (HPI)

H: all cells
PI: dead cells

Fig.

3:

Cytokine

profilescompared

between

Cetuximab-responsive and non-responsive PDOTS at day 3, measured fromFig. 1: Brightfield and HPI images of CRC spheroids embedded in matrix andspheroid supernatants. TNF⍺ was upregulated in 3/6 responsive

cultured in a singular channel of a microfluidic device.

PDOTS. IL-1β and IL-6 were elevated in non-responsive

Live tumor samples collected from patients are dissociated into

compared with responsive PDOTS samples.

spheroids within 24 hours of resection using a mechanical andfraction at baseline.

Experimental design
PDOTS isolated from n=32 CRC patient tumor specimens were used
to evaluate the therapeutic efficacy of Cetuximab. KRAS mutations
were determined pre-treatment by high-resolution melting analysis.
PDOTS were treated with Cetuximab or isotype-matched IgG1 control(300 µg/mL). On day 3 post-treatment, cytotoxicity was measured
using Hoechst/Propidium Iodide staining. Automated image analysis

Dendritic celllicensingcell profile was determined using flow cytometry on single cell S3

p=0.2

expressed transcripts from Nanostring panel. Differentiallyexpressed genes in treatment-responders relative to negative
control are associated with immune cell activity. For everytranscript, -log10(p-value) was plotted against the meanlog2(fold-change) between responsive and non-responsive

ADCCactivatedmaintain the patients’ native tumor microenvironment (TME). Tumor

Delta Area
Weighted %Deadmicrofluidic devices, mixed with a matrix with complete medium top=0.1

Fig. 7: Volcano plot representing statistically significantsamples.
Genes (top-right):
CD45RB, SOCS1, TRAF1, ICOS, TNFRSF18, GZMM, CASP1, SLAMF7, SH2DIA, PTPRC,
CD69, ICAM5, SERPINA1, REN, CD7, UBA7, TREM1, TNFSF8, FCGR2A, APOL6, PALMD,IRF4, WNT2B

Immunecellactivatedspheroids (S2 or PDOTS) 40 µm - 100 µm diameter are loaded onto

p=0.05

Evidence of immune cell activation

enzymatic digestion process. Samples are fractionated into threedifferent sizes: S1, S2 and S3 fractions. Patient derived organotypic-Log10(p-value)

p=0.01

Conclusions
Cetuximab non-responsive

Cetuximab responsive

Data generated from Xsphera’s platform are consistent with

Genes from top to bottom (Note: 2 patients with no GE data recorded were excluded from the set)
DC licensing: HLA-C, HLA-A, CD80, IL2, CD86, CD8A, CD40LG, CD27, CD70, CD28, IFNG,GZMB, HLA-DQA2, HLA-DQB1, CD8B, A2M, TLR4, HLA-DPB1, HLA-DPA1, CD40, CD4, IL4,PRF1, HLA-DQA1, HLA-DRA, HLA-B
ADCC-activated: TNF, KIR3DL2, NCR1, IFNG, GZMB, IL21R, XCL1/2, CCL3/L1, KIR3DL1,CCL4, KIR2DL3
Immune cell-activated: C2, TNFSF13, SH2D1A, NKG7, KLRD1, CD7, FCGR1A, CCL19, GAS1,CSF1, CSF2RB

clinical findings from Cetuximab response in CRC patients andPDOTS using PanCancer IO360 panel on nCounter® SPRINT ProfilerFig. 4: Heatmaps for specific pathways for Cetuximab-responsive

our data. Our findings on upregulation of DC-licensing and(Nanostring Technologies).

and non-responsive PDOTS at day 3 (Delta norm count shown).

activation of ADCC indicate involvement of immune cells in

Supported by NIH SBIR Grant 1 R43 CA261382-01A1
1Jenkins RW, Aref AR, et al. Ex Vivo Profiling of PD-1 Blockade Using Organotypic Tumor
Spheroids. Cancer Discov. 2018 Feb;8(2):196-215.
2De Roock W, Piessevaux H, et al. KRAS wild-type state predicts survival and is associatedto early radiological response in metastatic colorectal cancer treated with cetuximab. Ann
Oncol. 2008 Mar;19(3):508-15.
3Gelfo V, Mazzeschi M, et al. A Novel Role for the Interleukin-1 Receptor Axis in Resistanceto Anti-EGFR Therapy. Cancers. 2018, 10, 355.

Samples from the treatment-responsive group displayed elevatedcytotoxic response of CRC tumor cells to Cetuximab. Theselevels of genes pertaining to activation of ADCC. Additionally, weinsights could guide the development of more personalizedmeasured an increase in expression of genes responsible in thetherapeutic strategies, potentially improving the effectiveness ofprocess of dendritic cell licensing and immune cell activation.

treatments like Cetuximab.

was used to report cytotoxicity measurements. Cytokines secreted byspheroids were collected at day 3 and measured using Ella
(ProteinSimple, Biotechne). Bulk gene expression analysis wasperformed on purified RNA isolates derived from post-treatment

support evidence of Cetuximab-mediated suppression of EGFR
downstream signaling pathways. IL-1β has been reported toimpair the therapeutic efficacy of Cetuximab3, which aligns with