Spatial deconvolution without reference scRNA-seq

This is a tutorial on an example real Spatial Transcriptomics (ST) data (CID44971_TNBC) from Wu et al., 2021. Raw tutorial could be found in https://starfysh.readthedocs.io/en/latest/notebooks/Starfysh_tutorial_real.html

Starfysh performs cell-type deconvolution followed by various downstream analyses to discover spatial interactions in tumor microenvironment. Specifically, Starfysh looks for anchor spots (presumably with the highest compositions of one given cell type) informed by user-provided gene signatures (see example) as priors to guide the deconvolution inference, which further enables downstream analyses such as sample integration, spatial hub characterization, cell-cell interactions, etc. This tutorial focuses on the deconvolution task. Overall, Starfysh provides the following options:

At omicverse, we have made the following improvements:

• Easier visualization, you can use omicverse unified visualization for scientific mapping

• Reduce installation dependency errors, we optimized the automatic selection of different packages, you don’t need to install too many extra packages and cause conflicts.

Base feature:

• Spot-level deconvolution with expected cell types and corresponding annotated signature gene sets (default)

He, S., Jin, Y., Nazaret, A. et al. Starfysh integrates spatial transcriptomic and histologic data to reveal heterogeneous tumor–immune hubs. Nat Biotechnol (2024). https://doi.org/10.1038/s41587-024-02173-8

import cv2
import scanpy as sc
import omicverse as ov
ov.style(font_path='Arial')

🔬 Starting plot initialization...
Using already downloaded Arial font from: /tmp/omicverse_arial.ttf
Registered as: Arial
🧬 Detecting GPU devices…
✅ NVIDIA CUDA GPUs detected: 1
    • [CUDA 0] NVIDIA H100 80GB HBM3
      Memory: 79.1 GB | Compute: 9.0

   ____            _     _    __                  
  / __ \____ ___  (_)___| |  / /__  _____________ 
 / / / / __ `__ \/ / ___/ | / / _ \/ ___/ ___/ _ \ 
/ /_/ / / / / / / / /__ | |/ /  __/ /  (__  )  __/ 
\____/_/ /_/ /_/_/\___/ |___/\___/_/  /____/\___/                                              

🔖 Version: 1.7.9rc1   📚 Tutorials: https://omicverse.readthedocs.io/
✅ plot_set complete.

Step 1: Prepare spatial transcriptomics (1 min)

Purpose: load 10x Visium (Space Ranger outputs) or similar to obtain a coordinate-aware spatial AnnData (adata_sp).

• Inputs: Visium count matrix and spatial coordinates (from the spatial folder)

• Outputs: AnnData object (adata_sp) with spot coordinates and counts

• Key points:

  • Ensure maximal gene overlap with the scRNA-seq reference; map gene IDs if necessary.

  • For multiple samples, keep batch labels explicit to support merging and visualization.

adata_sp = sc.datasets.visium_sge(sample_id="V1_Human_Lymph_Node")
adata_sp.obs['sample'] = list(adata_sp.uns['spatial'].keys())[0]
adata_sp.var_names_make_unique()

reading /scratch/users/steorra/analysis/omic_test/data/V1_Human_Lymph_Node/filtered_feature_bc_matrix.h5
 (0:00:00)

Step 2: Prepare the gene sig marker

gene_sig means the dataframe stored the marker gene in each columns. If you don’t have it, you can calculated it using ov.space.calculate_gene_signature

# Load your scRNA-seq reference
# Example: Human lymph node reference
adata_sc = ov.datasets.sc_ref_Lymph_Node()
gene_sig=ov.space.calculate_gene_signature(
    adata_sc,
    clustertype='Subset',      # cell type name
    rank=True,
    key='rank_genes_groups',
    foldchange=2,
    topgenenumber=20    
)
gene_sig.head()

🧬 Loading SC reference data for Lymph Node
🔍 Downloading data to ./data/sc_ref_Lymph_Node.h5ad
⚠️ File ./data/sc_ref_Lymph_Node.h5ad already exists
 Loading data from ./data/sc_ref_Lymph_Node.h5ad
✅ Successfully loaded: 73260 cells × 10237 genes
...get cell type marker
ranking genes
WARNING: It seems you use rank_genes_groups on the raw count data. Please logarithmize your data before calling rank_genes_groups.
    finished: added to `.uns['rank_genes_groups']`
    'names', sorted np.recarray to be indexed by group ids
    'scores', sorted np.recarray to be indexed by group ids
    'logfoldchanges', sorted np.recarray to be indexed by group ids
    'pvals', sorted np.recarray to be indexed by group ids
    'pvals_adj', sorted np.recarray to be indexed by group ids (0:01:00)

	B_Cycling	B_GC_DZ	B_GC_LZ	B_GC_prePB	B_IFN	B_activated	B_mem	B_naive	B_plasma	B_preGC	...	T_CD4+_TfH	T_CD4+_TfH_GC	T_CD4+_naive	T_CD8+_CD161+	T_CD8+_cytotoxic	T_CD8+_naive	T_TIM3+	T_TfR	T_Treg	VSMC
0	HMGN2	CD79B	CD22	BIK	HLA-DPA1	HLA-DPA1	HLA-DPA1	HLA-DPA1	SDF2L1	HLA-DPA1	...	TRAC	TCF7	RPS15A	ZFP36	CD3E	NOSIP	TMSB4X	CD4	TRAC	TAGLN
1	DEK	TCEA1	PRPSAP2	PRPSAP2	ISG15	HVCN1	CD52	HVCN1	SSR3	CD72	...	CD69	SRGN	NOSIP	SRGN	CST7	TCF7	LCK	SRGN	SRGN	LGALS3
2	HMGN1	GAPDH	HMGN1	VPREB3	STAT1	CD74	SMIM14	CD79B	PPIB	NME2	...	SRGN	PASK	TCF7	RGCC	GZMK	RGCC	SRGN	CD3E	RGCC	CST3
3	GAPDH	SUGCT	GAPDH	HMCES	CD74	TCL1A	CD74	CD72	SEC61G	CD74	...	CD3E	ITM2A	RPS14	CD8A	NKG7	DNAJB1	CD3E	ZAP70	DNAJB1	LAPTM4A
4	HMGB1	EZR	CD74	MEF2B	IFIT3	HLA-DPB1	VPREB3	CD74	HERPUD1	HSP90AB1	...	SLC2A3	CD3E	LEF1	CCL5	CTSW	LEF1	CD27	TCRA_VDJsum	FOXP3	IGFBP5

5 rows × 34 columns

gene_sig.to_csv('data/gene_sig_Lymph_Node.csv')

gene_sig=ov.pd.read_csv('data/gene_sig_Lymph_Node.csv',index_col=0)
gene_sig.head()

	B_Cycling	B_GC_DZ	B_GC_LZ	B_GC_prePB	B_IFN	B_activated	B_mem	B_naive	B_plasma	B_preGC	...	T_CD4+_TfH	T_CD4+_TfH_GC	T_CD4+_naive	T_CD8+_CD161+	T_CD8+_cytotoxic	T_CD8+_naive	T_TIM3+	T_TfR	T_Treg	VSMC
0	HMGN2	CD79B	CD22	BIK	HLA-DPA1	HLA-DPA1	HLA-DPA1	HLA-DPA1	SDF2L1	HLA-DPA1	...	TRAC	TCF7	RPS15A	ZFP36	CD3E	NOSIP	TMSB4X	CD4	TRAC	TAGLN
1	DEK	TCEA1	PRPSAP2	PRPSAP2	ISG15	HVCN1	CD52	HVCN1	SSR3	CD72	...	CD69	SRGN	NOSIP	SRGN	CST7	TCF7	LCK	SRGN	SRGN	LGALS3
2	HMGN1	GAPDH	HMGN1	VPREB3	STAT1	CD74	SMIM14	CD79B	PPIB	NME2	...	SRGN	PASK	TCF7	RGCC	GZMK	RGCC	SRGN	CD3E	RGCC	CST3
3	GAPDH	SUGCT	GAPDH	HMCES	CD74	TCL1A	CD74	CD72	SEC61G	CD74	...	CD3E	ITM2A	RPS14	CD8A	NKG7	DNAJB1	CD3E	ZAP70	DNAJB1	LAPTM4A
4	HMGB1	EZR	CD74	MEF2B	IFIT3	HLA-DPB1	VPREB3	CD74	HERPUD1	HSP90AB1	...	SLC2A3	CD3E	LEF1	CCL5	CTSW	LEF1	CD27	TCRA_VDJsum	FOXP3	IGFBP5

5 rows × 34 columns

Step 3: Deconvolution with starfysh

We perform n_repeat random restarts and select the best model with lowest loss for parameter c (inferred cell-type proportions):

Starfysh is integrated into the omicverse.space.Deconvolution class. Simply set method='starfysh'.

Key Parameters

• n_repeats: Number of restart to run Starfysh.

• epochs: number of iterations

• device: the trainning device

# Initialize the Deconvolution object
decov_obj = ov.space.Deconvolution(
    adata_sp=adata_sp
)

decov_obj.preprocess_sp(
    mode='pearsonr',n_svgs=3000,target_sum=1e4,
    subset_genes=False,
)

🔍 [2026-01-11 23:07:13] Running preprocessing in 'cpu' mode...
Begin robust gene identification
    After filtration, 25187/36601 genes are kept.
    Among 25187 genes, 22411 genes are robust.
✅ Robust gene identification completed successfully.
Begin size normalization: shiftlog and HVGs selection pearson

🔍 Count Normalization:
   Target sum: 10000.0
   Exclude highly expressed: True
   Max fraction threshold: 0.2
   ⚠️ Excluding 1 highly-expressed genes from normalization computation
   Excluded genes: ['IGKC']

✅ Count Normalization Completed Successfully!
   ✓ Processed: 4,035 cells × 22,411 genes
   ✓ Runtime: 0.47s

🔍 Highly Variable Genes Selection (Experimental):
   Method: pearson_residuals
   Target genes: 3,000
   Theta (overdispersion): 100

✅ Experimental HVG Selection Completed Successfully!
   ✓ Selected: 3,000 highly variable genes out of 22,411 total (13.4%)
   ✓ Results added to AnnData object:
     • 'highly_variable': Boolean vector (adata.var)
     • 'highly_variable_rank': Float vector (adata.var)
     • 'highly_variable_nbatches': Int vector (adata.var)
     • 'highly_variable_intersection': Boolean vector (adata.var)
     • 'means': Float vector (adata.var)
     • 'variances': Float vector (adata.var)
     • 'residual_variances': Float vector (adata.var)
    Time to analyze data in cpu: 3.05 seconds.
computing PCA
    with n_comps=50
    finished (0:00:01)
✅ Preprocessing completed successfully.
    Added:
        'highly_variable_features', boolean vector (adata.var)
        'means', float vector (adata.var)
        'variances', float vector (adata.var)
        'residual_variances', float vector (adata.var)
        'counts', raw counts layer (adata.layers)
    End of size normalization: shiftlog and HVGs selection pearson
✓ spatial transcriptomics data is preprocessed

%time
# Run FlashDeconv deconvolution
decov_obj.deconvolution(
    method='starfysh',
    gene_sig=gene_sig,  
    starfysh_kwargs={
        'n_repeats':3,
        'epochs':200,
        'patience':50,
        'device':None,
        'batch_size':32,
        'alpha_mul':50,
        'lr':1e-4,
        'poe':False,
        'verbose':True,
        'n_anchors':60,
        'window_size':3,
    }
)

CPU times: user 3 μs, sys: 0 ns, total: 3 μs
Wall time: 7.87 μs
30 components are retained using conditional_number=40.00
Epoch[10/200], train_loss: 1964.5555, train_reconst: 1717.3358, train_u: 17.7731,train_z: 12.2491,train_c: 216.1686,train_l: 1.0289
Epoch[20/200], train_loss: 1836.5867, train_reconst: 1631.5397, train_u: 17.3562,train_z: 8.2599,train_c: 177.4633,train_l: 1.9675
Epoch[30/200], train_loss: 1803.6228, train_reconst: 1611.8570, train_u: 17.0200,train_z: 6.8935,train_c: 165.4288,train_l: 2.4234
Epoch[40/200], train_loss: 1784.1136, train_reconst: 1599.9807, train_u: 16.7433,train_z: 6.1974,train_c: 158.4979,train_l: 2.6943
Epoch[50/200], train_loss: 1771.6689, train_reconst: 1591.6377, train_u: 16.5159,train_z: 5.8556,train_c: 154.8143,train_l: 2.8453
Epoch[60/200], train_loss: 1761.9189, train_reconst: 1585.5430, train_u: 16.3333,train_z: 5.7242,train_c: 151.3896,train_l: 2.9288
Epoch[70/200], train_loss: 1755.7707, train_reconst: 1580.6499, train_u: 16.1848,train_z: 5.6212,train_c: 150.3357,train_l: 2.9791
Epoch[80/200], train_loss: 1748.3032, train_reconst: 1575.9084, train_u: 16.0641,train_z: 5.6020,train_c: 147.7620,train_l: 2.9667
Epoch[90/200], train_loss: 1745.4689, train_reconst: 1573.8276, train_u: 15.9672,train_z: 5.5890,train_c: 147.1002,train_l: 2.9849
Epoch[100/200], train_loss: 1741.4059, train_reconst: 1571.2467, train_u: 15.8885,train_z: 5.5138,train_c: 145.7818,train_l: 2.9751
Epoch[110/200], train_loss: 1739.2005, train_reconst: 1569.5755, train_u: 15.8249,train_z: 5.5090,train_c: 145.3119,train_l: 2.9792
Epoch[120/200], train_loss: 1737.3613, train_reconst: 1568.0982, train_u: 15.7730,train_z: 5.5225,train_c: 144.9962,train_l: 2.9714
Epoch[130/200], train_loss: 1735.5456, train_reconst: 1567.2170, train_u: 15.7308,train_z: 5.5234,train_c: 144.1060,train_l: 2.9684
Epoch[140/200], train_loss: 1733.8153, train_reconst: 1565.6691, train_u: 15.6964,train_z: 5.5186,train_c: 143.9690,train_l: 2.9622
Epoch[150/200], train_loss: 1734.0409, train_reconst: 1565.6646, train_u: 15.6686,train_z: 5.5101,train_c: 144.2291,train_l: 2.9684
Epoch[160/200], train_loss: 1733.5203, train_reconst: 1564.8707, train_u: 15.6460,train_z: 5.5183,train_c: 144.5110,train_l: 2.9744
Epoch[170/200], train_loss: 1731.6396, train_reconst: 1563.7964, train_u: 15.6274,train_z: 5.5189,train_c: 143.7256,train_l: 2.9713
Epoch[180/200], train_loss: 1730.6814, train_reconst: 1563.4632, train_u: 15.6123,train_z: 5.4934,train_c: 143.1347,train_l: 2.9779
Epoch[190/200], train_loss: 1730.8846, train_reconst: 1563.2024, train_u: 15.6000,train_z: 5.5424,train_c: 143.5656,train_l: 2.9743
Epoch[200/200], train_loss: 1730.3862, train_reconst: 1563.3851, train_u: 15.5899,train_z: 5.4530,train_c: 142.9817,train_l: 2.9766
Epoch[10/200], train_loss: 1964.5555, train_reconst: 1717.3358, train_u: 17.7731,train_z: 12.2491,train_c: 216.1686,train_l: 1.0289
Epoch[20/200], train_loss: 1836.5867, train_reconst: 1631.5397, train_u: 17.3562,train_z: 8.2599,train_c: 177.4633,train_l: 1.9675
Epoch[30/200], train_loss: 1803.6228, train_reconst: 1611.8570, train_u: 17.0200,train_z: 6.8935,train_c: 165.4288,train_l: 2.4234
Epoch[40/200], train_loss: 1784.1136, train_reconst: 1599.9807, train_u: 16.7433,train_z: 6.1974,train_c: 158.4979,train_l: 2.6943
Epoch[50/200], train_loss: 1771.6689, train_reconst: 1591.6377, train_u: 16.5159,train_z: 5.8556,train_c: 154.8143,train_l: 2.8453
Epoch[60/200], train_loss: 1761.9189, train_reconst: 1585.5430, train_u: 16.3333,train_z: 5.7242,train_c: 151.3896,train_l: 2.9288
Epoch[70/200], train_loss: 1755.7707, train_reconst: 1580.6499, train_u: 16.1848,train_z: 5.6212,train_c: 150.3357,train_l: 2.9791
Epoch[80/200], train_loss: 1748.3032, train_reconst: 1575.9084, train_u: 16.0641,train_z: 5.6020,train_c: 147.7620,train_l: 2.9667
Epoch[90/200], train_loss: 1745.4689, train_reconst: 1573.8276, train_u: 15.9672,train_z: 5.5890,train_c: 147.1002,train_l: 2.9849
Epoch[100/200], train_loss: 1741.4059, train_reconst: 1571.2467, train_u: 15.8885,train_z: 5.5138,train_c: 145.7818,train_l: 2.9751
Epoch[110/200], train_loss: 1739.2005, train_reconst: 1569.5755, train_u: 15.8249,train_z: 5.5090,train_c: 145.3119,train_l: 2.9792
Epoch[120/200], train_loss: 1737.3613, train_reconst: 1568.0982, train_u: 15.7730,train_z: 5.5225,train_c: 144.9962,train_l: 2.9714
Epoch[130/200], train_loss: 1735.5456, train_reconst: 1567.2170, train_u: 15.7308,train_z: 5.5234,train_c: 144.1060,train_l: 2.9684
Epoch[140/200], train_loss: 1733.8153, train_reconst: 1565.6691, train_u: 15.6964,train_z: 5.5186,train_c: 143.9690,train_l: 2.9622
Epoch[150/200], train_loss: 1734.0409, train_reconst: 1565.6646, train_u: 15.6686,train_z: 5.5101,train_c: 144.2291,train_l: 2.9684
Epoch[160/200], train_loss: 1733.5203, train_reconst: 1564.8707, train_u: 15.6460,train_z: 5.5183,train_c: 144.5110,train_l: 2.9744
Epoch[170/200], train_loss: 1731.6396, train_reconst: 1563.7964, train_u: 15.6274,train_z: 5.5189,train_c: 143.7256,train_l: 2.9713
Epoch[180/200], train_loss: 1730.6814, train_reconst: 1563.4632, train_u: 15.6123,train_z: 5.4934,train_c: 143.1347,train_l: 2.9779
Epoch[190/200], train_loss: 1730.8846, train_reconst: 1563.2024, train_u: 15.6000,train_z: 5.5424,train_c: 143.5656,train_l: 2.9743
Epoch[200/200], train_loss: 1730.3862, train_reconst: 1563.3851, train_u: 15.5899,train_z: 5.4530,train_c: 142.9817,train_l: 2.9766
Epoch[10/200], train_loss: 1964.5555, train_reconst: 1717.3358, train_u: 17.7731,train_z: 12.2491,train_c: 216.1686,train_l: 1.0289
Epoch[20/200], train_loss: 1836.5867, train_reconst: 1631.5397, train_u: 17.3562,train_z: 8.2599,train_c: 177.4633,train_l: 1.9675
Epoch[30/200], train_loss: 1803.6228, train_reconst: 1611.8570, train_u: 17.0200,train_z: 6.8935,train_c: 165.4288,train_l: 2.4234
Epoch[40/200], train_loss: 1784.1136, train_reconst: 1599.9807, train_u: 16.7433,train_z: 6.1974,train_c: 158.4979,train_l: 2.6943
Epoch[50/200], train_loss: 1771.6689, train_reconst: 1591.6377, train_u: 16.5159,train_z: 5.8556,train_c: 154.8143,train_l: 2.8453
Epoch[60/200], train_loss: 1761.9189, train_reconst: 1585.5430, train_u: 16.3333,train_z: 5.7242,train_c: 151.3896,train_l: 2.9288
Epoch[70/200], train_loss: 1755.7707, train_reconst: 1580.6499, train_u: 16.1848,train_z: 5.6212,train_c: 150.3357,train_l: 2.9791
Epoch[80/200], train_loss: 1748.3032, train_reconst: 1575.9084, train_u: 16.0641,train_z: 5.6020,train_c: 147.7620,train_l: 2.9667
Epoch[90/200], train_loss: 1745.4689, train_reconst: 1573.8276, train_u: 15.9672,train_z: 5.5890,train_c: 147.1002,train_l: 2.9849
Epoch[100/200], train_loss: 1741.4059, train_reconst: 1571.2467, train_u: 15.8885,train_z: 5.5138,train_c: 145.7818,train_l: 2.9751
Epoch[110/200], train_loss: 1739.2005, train_reconst: 1569.5755, train_u: 15.8249,train_z: 5.5090,train_c: 145.3119,train_l: 2.9792
Epoch[120/200], train_loss: 1737.3613, train_reconst: 1568.0982, train_u: 15.7730,train_z: 5.5225,train_c: 144.9962,train_l: 2.9714
Epoch[130/200], train_loss: 1735.5456, train_reconst: 1567.2170, train_u: 15.7308,train_z: 5.5234,train_c: 144.1060,train_l: 2.9684
Epoch[140/200], train_loss: 1733.8153, train_reconst: 1565.6691, train_u: 15.6964,train_z: 5.5186,train_c: 143.9690,train_l: 2.9622
Epoch[150/200], train_loss: 1734.0409, train_reconst: 1565.6646, train_u: 15.6686,train_z: 5.5101,train_c: 144.2291,train_l: 2.9684
Epoch[160/200], train_loss: 1733.5203, train_reconst: 1564.8707, train_u: 15.6460,train_z: 5.5183,train_c: 144.5110,train_l: 2.9744
Epoch[170/200], train_loss: 1731.6396, train_reconst: 1563.7964, train_u: 15.6274,train_z: 5.5189,train_c: 143.7256,train_l: 2.9713
Epoch[180/200], train_loss: 1730.6814, train_reconst: 1563.4632, train_u: 15.6123,train_z: 5.4934,train_c: 143.1347,train_l: 2.9779
Epoch[190/200], train_loss: 1730.8846, train_reconst: 1563.2024, train_u: 15.6000,train_z: 5.5424,train_c: 143.5656,train_l: 2.9743
Epoch[200/200], train_loss: 1730.3862, train_reconst: 1563.3851, train_u: 15.5899,train_z: 5.4530,train_c: 142.9817,train_l: 2.9766
✓ starfysh is done
The starfysh result is saved in self.adata_cell2location
The starfysh model is saved in self.starfysh_model

Step 4: Visualization (5–15 min)

We provide multiple views: single-target spatial heatmaps, multi-target overlays, and local pie charts. Start with global inspection, then zoom into biologically relevant regions for higher-resolution assessment.

4.1 Spatial value dotplot

annotation_list=['B_Cycling', 'B_GC_LZ', 'T_CD4+_TfH_GC', 'FDC',
                'B_naive', 'T_CD4+_naive', 'B_plasma', 'Endo']
sc.pl.spatial(
    decov_obj.adata_cell2location, 
    cmap='magma',
    # show first 8 cell types
    color=annotation_list,
    ncols=4, size=1.3,
    img_key='hires',
    # limit color scale at 99.2% quantile of cell abundance
    #vmin=0, vmax='p99.2'
)

4.2 Multi-target overlay

import matplotlib as mpl
clust_labels = annotation_list[:5]
clust_col = ['' + str(i) for i in clust_labels] # in case column names differ from labels

with mpl.rc_context({'figure.figsize': (6, 6),'axes.grid': False}):
    fig = ov.pl.plot_spatial(
        adata=decov_obj.adata_cell2location,
        # labels to show on a plot
        color=clust_col, labels=clust_labels,
        show_img=True,
        # 'fast' (white background) or 'dark_background'
        style='fast',
        # limit color scale at 99.2% quantile of cell abundance
        max_color_quantile=0.992,
        # size of locations (adjust depending on figure size)
        circle_diameter=4,
        reorder_cmap = [#0,
            1,2,3,4,6], #['yellow', 'orange', 'blue', 'green', 'purple', 'grey', 'white'],
        colorbar_position='right',
        #palette=color_dict
    )
    

4.3 Pie plot

We recommend cropping a region of interest before plotting to avoid overly dense pie charts on whole slides.

adata_s = ov.space.crop_space_visium(
    decov_obj.adata_cell2location, 
    crop_loc=(0, 0),      
    crop_area=(500, 1000), 
    library_id=list(decov_obj.adata_cell2location.uns['spatial'].keys())[0] , 
    scale=1
)

sc.pl.spatial(adata_s, cmap='magma',
                  # show first 8 cell types
                  color=annotation_list[0],
                  ncols=4, size=1.3,
                  img_key='hires',
                  # limit color scale at 99.2% quantile of cell abundance
                  #vmin=0, vmax='p99.2'
                 )

color_dict=dict(zip(annotation_list,
                   ov.pl.sc_color))

fig, ax = ov.plt.subplots(figsize=(8, 4))
sc.pl.spatial(
    adata_s, 
    basis='spatial',
    color=None,  
    size=1.3,
    img_key='hires',
    ax=ax,      
    show=False
)

ov.pl.add_pie2spatial(
    adata_s,
    img_key='hires',
    cell_type_columns=annotation_list[:],
    ax=ax,
    colors=color_dict,
    pie_radius=10,
    remainder='gap',
    legend_loc=(0.5, -0.25),
    ncols=4,
    alpha=0.8
)

#plt.show()

<Axes: xlabel='spatial1', ylabel='spatial2'>