Patient-Specific Vascularized Tumor Model: Blocking TAM Recruitment with Multispecific Antibodies Targeting CCR2 and CSF-1R

Tumor-associated inflammation drives cancer progression and therapy resistance, with the infiltration of monocyte-derived tumor-associated macrophages (TAMs) associated with poor prognosis in diverse cancers. Targeting TAMs holds potential against solid tumors, but effective immunotherapies require testing on immunocompetent human models prior to clinical trials. Here, we develop an in vitro model of microvascular networks that incorporates tumor spheroids or patient tissues. By perfusing the vasculature with human monocytes, we investigate monocyte trafficking into the tumor and evaluate immunotherapies targeting the human tumor microenvironment. Our findings demonstrate that macrophages in vascularized breast and lung tumor models can enhance monocyte recruitment via TAM-produced CCL7 and CCL2, mediated by CSF-1R. Additionally, we assess a novel multispecific antibody targeting CCR2, CSF-1R, and neutralizing TGF-β, referred to as CSF1R/CCR2/TGF-β Ab, on monocytes and macrophages using our 3D models. This antibody repolarizes TAMs towards an anti-tumoral M1-like phenotype, reduces monocyte chemoattractant protein secretion, and effectively blocks monocyte migration. Finally, we show that the CSF1R/CCR2/TGF-β Ab inhibits monocyte recruitment in patient-specific vascularized tumor models. Overall, this vascularized tumor model offers valuable insights into monocyte recruitment and enables functional testing of innovative therapeutic antibodies targeting TAMs in the tumor microenvironment (TME).


Supplementary Figures
Figure S1: Step-by-step illustration of gel well formation using meniscus trapping mechanism.1

) Side, front and section views of an exemplary microfluidic device having 2 media channels and one gel channel with one central port showing the steps for gel loading to form a vascular bed having a central empty gel well. The gel solution is loaded into a pipette tip. The volume of the gel solution is approximately the volume of the central channel minus the volume of the central well. 2) Gel injection. The gel is
loaded into the device while tilting it to facilitate the gel confinement using the partial walls separating the gel channel and two media channels.3) Gel hole formation.The pipette tip is then removed and the device is gently tapped to cause the gel solution to advance toward the gel outlet on the right ensuring that the gel inside of the hole is evacuated.4) Gel polymerization on horizontal direction.The device is placed on a flat surface at 37°C inside an incubator until the central hole is dry and gelation occurs.The imbalance of surface tensions at the two interfaces (∆ 1 > ∆ 2 ), explains the removal of liquid from the central gel well since the pressure drop across the air-liquid interface at the media channel is greater than that inside the well.Media is prevented from entering into the well when media is later introduced into the media channel by the hydrophobic nature of fibrin gel.

Figure S12: Chemotaxis coefficient of TAMs in unidirectional migration assays in devices treated with TGF-β (TAM-TGFb) and not treated (TAM). Each point represents a ROI, data were pooled from several devices. Statistical significance is obtained with
Student's t-test; nf: non-significant.

Supplementary information S1. Methods for forming a dry central well
The hydrogel solution is introduced into the inlet and allowed to spread within the gel channel (Fig. S1.1).During injection, the device is tilted to promote the distribution of the gel within the channel.Initially, the gel fills the space beneath the central hole, gradually progressing further along the gel channel (Fig. S1.2).The gentle tapping of the device helps the gel solution reach the remaining volume of the chamber (Fig. S1.3).Consequently, the gel coats the inner surface of the gel channel and is retained within it due to capillary forces.
Once the channel's surface is wet, the hydrogel solution forms an angle with the hydrophilic glass slide, creating a curvature that confines the solution within a capillary chamber formed by the PDMS and glass slide.When the device is laid flat, gravity naturally tends to draw the gel solution back into the central hole (Fig. S1.4).However, this is counteracted by the surface tension established at the junction between the glass and the solution in the chamber.Therefore, the combination of capillary forces and the curvature resulting from surface tension generates a negative pressure that prevents the gel solution from escaping back into the central hole (see below).The interface at the central hole also possesses a curvature, but its radius is larger, making it incapable of pulling the solution back into the hole.
Once the gel solution solidifies, the fibrin surface becomes dry and hydrophobic.Consequently, when media is introduced into the media channel on the side, it cannot penetrate the central hole, keeping the entire central region dry throughout vascular formation.If there is a need to introduce tumor spheroids or patient tissues into the central hole, the surface tension at the gel-air interface needs to be disrupted.This can be achieved by wetting the central hole with media and then carefully transferring the tumoral tissue.The tissue can be allowed to sink from a pipette tip to the bottom of the central well by gravity.
The following outlines the procedure for calculating surface tension during and after gel loading.When the gel is introduced into the channel and comes into contact with the channel's surface, capillary forces trap the gel solution between the glass and the PDMS layer.This trapping phenomenon arises due to an imbalance in surface tensions at the two interfaces (as explained below).
At the interface between the gel channel and the media channel, the Laplace pressure, representing the difference between the external and internal pressures, is determined.This pressure is expressed as: Where r denotes the curvature radius of the air-liquid interface between the gel and the media channel,  is the surface tension of the fibrin gel solution.
Similarly the pressure across the interface between the gel channel and the central hole is given by: Once the device is placed horizontally (as shown in Fig. S2, Step 4), the gel solution inside the channel reaches equilibrium.Provided ∆ 1 > ∆ 2 a pressure difference exists to drain fluid from the central hole.Initially, the hole forms a spherical concave well where  1 and  2 are approximately the radius of the central hole and  ≅ 1 since  is approximately zero while the hole is being formed, leaving behind a thin liquid layer.Additionally, we have the relationship

𝑟 = ℎ 𝑐𝑜𝑠𝛿
With ℎ is the height of the gel region (0.5mm) minus the thickness of the partial wall (0.166 mm),  is the contact angle at the interface between the gel solution and media channel.Thus, Given that  ℎ = 0.75 , ℎ = 0.33 ,  ≅ 1 as the meniscus recedes from the wetted area, the following imbalance in pressure drop occurs immediately after injection: This imbalance effectively retains the gel solution inside the gel chamber, preventing it from re-entering the central hole.
After fibrin gel solidifies, the fibrin-air interface becomes hydrophobic.This hydrophobicity hinders the infiltration of media from the media channel into the gel and the central hole, as further discussed in this publication 1 .

S2. Diffusion and chemotaxis coefficient calculation
To compare monocyte chemotaxis in different conditions, we apply the Keller and Segel (1971)  During the experiment (0-3 days) we assume that monocyte death is negligible and, according to previously published results, that monocytes exhibit little proliferation 4 , therefore: Consistent with other studies we, too, assume that the random mobility coefficient is independent of chemotractant concentration, so () =  = constant.We verified this assumption in the main text (Fig. 3Ci).This will be assumed to be the case for our calculations below, unless otherwise specified.These equations and assumptions are reviewed by Tindall et al 5 .We thus obtain the following equation: Similarly, from the continuity equation of chemokines in the condition in which there is no convection as in our experiments, we obtain: Where (, ) represents chemoattractant degradation due to either cell consumption or chemical reaction and   the diffusion coefficient of solute .
Under steady state conditions   = 0, and we assume that chemotractant consumption is negligible g(, ) ≅ 0, we can write: We further assume that n = n(x) in our 1-D experiments and apply the cell and chemoattractant conservation equations (E.S2) and (E.S3) to the single channel device (Fig. S2), obtaining: This is the governing continuity equation for chemoattractant.We define a chemotaxis coefficient: We next develop a method based on this analysis for estimating the chemotaxis coefficient from the cell concentration profile within the gel channel of our single-channel device from day 0 to day 2 after perfusing monocytes into one media channel.We divide the device into 4 compartments (Fig S2): the two media channels 1 and 4, compartment 2 and 3 have the same width and constitute the gel channel, separated by cross-section B. The chemoattractant source is introduced into channel 1 and monocytes are introduced into channel 4. EC monolayer is grown at the interface between compartment 3 and channel 4.
We image the device daily and compare the cell distribution to the migration profile simulation.The total numbers of cells in compartments 2 and 3 are the integral of the function  3 = ∫  Integration between day 1 and day 2:

Figure S2 :
Figure S2: Schematic illustration of a single gel channel device with an endothelial monolayer for unidirectional monocyte migration assays MDA-MB-231 TFM (231 TFM) spheroids.C) Gating of dead and live cells using Zombie Green TM .D) Quantification of overall cell dead in various triculture conditions.From left to right: 231 TFM, 231 TFM treated with BLZ945 drug, anti-CSF-1R antibody, or CSF1R/CCR2/TGF-β Ab, MDA-MB-468, A-427, A-594 and H2009 TFM spheroids.Each point represents the data obtained by one dissociated spheroid.

Figure S5 :
Figure S5: Diagram showing a quantification method based on vasculature (green) and tumor spheroid (orange).Immune cells flowing inside the vascular networks, extravasate and migrate toward the tumor spheroid.They can be regrouped into 3 categories: (1) immune cells that extravasated and migrated toward the tumor spheroid are the ones that are inside the center volume below the hole and infiltrate the tumor spheroid; (2) Immune cells that extravasate but did not move toward the tumor spheroid and; (3) immune cells that stay luminal.Percentage of monocytes in different locations relative to the vasculatures.Percentage of cells= # cells in different compartments/ total cell #.Monocytes can stay inside the vasculatures (Luminal) or outside the vasculatures (Extravasated, including migrated and not migrated cells).The recruited cells are those extravasated and migrated into the hole containing the tumor spheroid.

Figure S9 :
Figure S9: Representative flow cytometry histogram showing the level of chemokine receptor CXCR2 (top) and CCR2 (bottom) expressions of monocytes compared to isotype controls.

Figure S10 :
Figure S10: Percentage of transmigrated monocytes in transwells with or without an EC monolayer.Statistical significance is obtained with Student's t-test; ****<0.0001

Figure S11 :
Figure S11: CD206 expression on original bone-marrow derived monocytes, denoted as "original", or monocytesthat perform migration from upper to lower transwell with or without the presence of endothelial monolayer, denoted as "with EC" or "without EC", respectively.From left to right, first 3 figures: selection of live monocytes using several gating strategies based on size and Zombie Green TM staining, 4 th figure: monocyte expression of CD206.

Figure S13 :
Figure S13: Monocytes become bigger and less round, more elongated over 5 days after extravasation in a device with a 231 TF spheroid.
model of chemotaxis on the geometry of our devices and calculate the chemotaxis coefficients of each condition 2 .First, we write the equation for the conservation of cell number: (, )  + .  + . ℎ =   −  ℎ where (, ) is the concentration of cells. ℎ = () represents cell chemotaxis where () the intrinsic chemotaxis coefficient,  the concentration of the chemokine, and the diffusional cell flux is defined as   = −() with () the diffusion coefficient of the cells due to their random, non-directed motion.In general, () can depend on  due to chemokinesis 3 .Finally, we represent the cell proliferation rate by  + and cell death rate by  − , thereby obtaining the generalized form of the Keller and Segel (1971) model of chemotaxis:   = ( + −  − ) − .() + .() Equation (E.S1)